Modulation of Factor 11 Expression

ABSTRACT

Disclosed herein are antisense compounds and methods for decreasing Factor 11 and treating or preventing thromboembolic complications in an individual in need thereof. Examples of disease conditions that can be ameliorated with the administration of antisense compounds targeted to Factor 11 include thrombosis, embolism, and thromboembolism, such as, deep vein thrombosis, pulmonary embolism, myocardial infarction, and stroke. Antisense compounds targeting Factor 11 can also be used as a prophylactic treatment to prevent individuals at risk for thrombosis and embolism.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims priority under 35 U.S.C. §119(e) to U.S. Provisional Application No. 61/105,772, filed Oct. 15, 2008 and U.S. Provisional Application No. 61/174,461, filed Apr. 30, 2009. Each of the above applications is herein incorporated by reference in its entirety.

SEQUENCE LISTING

The present application is being filed along with a Sequence Listing in electronic format. The Sequence Listing is provided as a file entitled 20091015_BIOL0107USSEQ.txt created Oct. 15, 2009, which is 92 Kb in size. The information in the electronic format of the sequence listing is incorporated herein by reference in its entirety.

FIELD OF THE INVENTION

Embodiments of the present invention provide methods, compounds, and compositions for reducing expression of Factor 11 mRNA and protein in an animal. Such methods, compounds, and compositions are useful to treat, prevent, or ameliorate thromboembolic complications.

BACKGROUND OF THE INVENTION

The circulatory system requires mechanisms that prevent blood loss, as well as those that counteract inappropriate intravascular obstructions. Generally, coagulation comprises a cascade of reactions culminating in the conversion of soluble fibrinogen to an insoluble fibrin gel. The steps of the cascade involve the conversion of an inactive zymogen to an activated enzyme. The active enzyme then catalyzes the next step in the cascade.

Coagulation Cascade

The coagulation cascade may be initiated through two branches, the tissue factor pathway (also “extrinsic pathway”), which is the primary pathway, and the contact activation pathway (also “intrinsic pathway”).

The tissue factor pathway is initiated by the cell surface receptor tissue factor (TF, also referred to as factor III), which is expressed constitutively by extravascular cells (pericytes, cardiomyocytes, smooth muscle cells, and keratinocytes) and expressed by vascular monocytes and endothelial cells upon induction by inflammatory cytokines or endotoxin. (Drake et al., Am J Pathol 1989, 134:1087-1097). TF is the high affinity cellular receptor for coagulation factor VIIa, a serine protease. In the absence of TF, VIIa has very low catalytic activity, and binding to TF is necessary to render VIIa functional through an allosteric mechanism. (Drake et al., Am J Pathol 1989, 134:1087-1097). The TF-VIIa complex activates factor X to Xa. Xa in turn associates with its co-factor factor Va into a prothrombinase complex which in turn activates prothrombin, (also known as factor II or factor 2) to thrombin (also known as factor IIa, or factor 2a). Thrombin activates platelets, converts fibrinogen to fibrin and promotes fibrin cross-linking by activating factor XIII, thus forming a stable plug at sites where TF is exposed on extravascular cells. In addition, thrombin reinforces the coagulation cascade response by activating factors V and VIII.

The contact activation pathway is triggered by activation of factor XII to XIIa. Factor XIIa converts XI to XIa, and XIa converts IX to IXa. IXa associates with its cofactor VIIIa to convert X to Xa. The two pathways converge at this point as factor Xa associates factor Va to activate prothrombin (factor II) to thrombin (factor IIa).

Inhibition of Coagulation.

At least three mechanisms keep the coagulation cascade in check, namely the action of activated protein C, antithrombin, and tissue factor pathway inhibitor. Activated protein C is a serine protease that degrades cofactors Va and VIIIa. Protein C is activated by thrombin with thrombomodulin, and requires coenzyme Protein S to function. Antithrombin is a serine protease inhibitor (serpin) that inhibits serine proteases: thrombin, Xa, XIIa, XIa and IXa. Tissue factor pathway inhibitor inhibits the action of Xa and the TF-VIIa complex. (Schwartz A L et al., Trends Cardiovasc Med. 1997; 7:234-239.)

Disease

Thrombosis is the pathological development of blood clots, and an embolism occurs when a blood clot migrates to another part of the body and interferes with organ function. Thromboembolism may cause conditions such as deep vein thrombosis, pulmonary embolism, myocardial infarction, and stroke. Significantly, thromboembolism is a major cause of morbidity affecting over 2 million Americans every year. (Adcock et al. American Journal of Clinical Pathology. 1997; 108:434-49). While most cases of thrombosis are due to acquired extrinsic problems, for example, surgery, cancer, immobility, some cases are due to a genetic predisposition, for example, antiphospholipid syndrome and the autosomal dominant condition, Factor V Leiden. (Bertina R M et al. Nature 1994; 369:64-67.)

Treatment.

The most commonly used anticoagulants, warfarin, heparin, and low molecular weight heparin (LMWH) all possess significant drawbacks.

Warfarin is typically used to treat patients suffering from atrial fibrillation. The drug interacts with vitamin K-dependent coagulation factors which include factors II, VII, IX and X. Anticoagulant proteins C and S are also inhibited by warfarin. Drug therapy using warfarin is further complicated by the fact that warfarin interacts with other medications, including drugs used to treat atrial fibrillation, such as amiodarone. Because therapy with warfarin is difficult to predict, patients must be carefully monitored in order to detect any signs of anomalous bleeding.

Heparin functions by activating antithrombin which inhibits both thrombin and factor X. (Bjork I, Lindahl U. Mol Cell Biochem. 1982 48: 161-182.) Treatment with heparin may cause an immunological reaction that makes platelets aggregate within blood vessels that can lead to thrombosis. This side effect is known as heparin-induced thrombocytopenia (HIT) and requires patient monitoring. Prolonged treatment with heparin may also lead to osteoporosis. LMWH can also inhibit Factor 2, but to a lesser degree than unfractioned heparin (UFH). LMWH has been implicated in the development of HIT.

Thus, current anticoagulant agents lack predictability and specificity and, therefore, require careful patient monitoring to prevent adverse side effects, such as bleeding complications. There are currently no anticoagulants which target only the intrinsic or extrinsic pathway.

SUMMARY OF THE INVENTION

Provided herein are methods, compounds, and compositions for modulating expression of Factor 11 mRNA and protein. In certain embodiments, Factor 11 specific inhibitors modulate expression of Factor 11 mRNA and protein. In certain embodiments, Factor 11 specific inhibitors are nucleic acids, proteins, or small molecules.

In certain embodiments, modulation can occur in a cell or tissue. In certain embodiments, the cell or tissue is in an animal. In certain embodiments, the animal is a human. In certain embodiments, Factor 11 mRNA levels are reduced. In certain embodiments, Factor 11 protein levels are reduced. Such reduction can occur in a time-dependent manner or in a dose-dependent manner.

Also provided are methods, compounds, and compositions useful for preventing, treating, and ameliorating diseases, disorders, and conditions. In certain embodiments, such diseases, disorders, and conditions are thromboembolic complications. Such thromboembolic complications include the categories of thrombosis, embolism, and thromboembolism. In certain embodiments such thromboembolic complications include deep vein thrombosis, pulmonary embolism, myocardial infarction, and stroke.

Such diseases, disorders, and conditions can have one or more risk factors, causes, or outcomes in common. Certain risk factors and causes for development of a thromboembolic complication include immobility, surgery (particularly orthopedic surgery), malignancy, pregnancy, older age, use of oral contraceptives, atrial fibrillation, previous thromboembolic complication, chronic inflammatory disease, and inherited or acquired prothrombotic clotting disorders. Certain outcomes associated with development of a thromboembolic complication include decreased blood flow through an affected vessel, death of tissue, and death.

In certain embodiments, methods of treatment include administering a Factor 11 specific inhibitor to an individual in need thereof.

DETAILED DESCRIPTION OF THE INVENTION

It is to be understood that both the foregoing general description and the following detailed description are exemplary and explanatory only and are not restrictive of the invention, as claimed. Herein, the use of the singular includes the plural unless specifically stated otherwise. As used herein, the use of “or” means “and/or” unless stated otherwise. Furthermore, the use of the term “including” as well as other forms, such as “includes” and “included”, is not limiting. Also, terms such as “element” or “component” encompass both elements and components comprising one unit and elements and components that comprise more than one subunit, unless specifically stated otherwise.

The section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described. All documents, or portions of documents, cited in this application, including, but not limited to, patents, patent applications, articles, books, and treatises, are hereby expressly incorporated by reference for the portions of the document discussed herein, as well as in their entirety.

DEFINITIONS

Unless specific definitions are provided, the nomenclature utilized in connection with, and the procedures and techniques of, analytical chemistry, synthetic organic chemistry, and medicinal and pharmaceutical chemistry described herein are those well known and commonly used in the art.

Standard techniques may be used for chemical synthesis, and chemical analysis. Where permitted, all patents, applications, published applications and other publications, GENBANK Accession Numbers and associated sequence information obtainable through databases such as National Center for Biotechnology Information (NCBI) and other data referred to throughout in the disclosure herein are incorporated by reference for the portions of the document discussed herein, as well as in their entirety.

Unless otherwise indicated, the following terms have the following meanings:

“2′-O-methoxyethyl” (also 2′-MOE and 2′-O(CH₂)₂—OCH₃) refers to an O-methoxy-ethyl modification of the 2′ position of a furosyl ring. A 2′-O-methoxyethyl modified sugar is a modified sugar.

“2′-O-methoxyethyl nucleotide” means a nucleotide comprising a 2′-O-methoxyethyl modified sugar moiety.

“5-methylcytosine” means a cytosine modified with a methyl group attached to the 5′ position. A 5-methylcytosine is a modified nucleobase.

“Active pharmaceutical agent” means the substance or substances in a pharmaceutical composition that provide a therapeutic benefit when administered to an individual. For example, in certain embodiments an antisense oligonucleotide targeted to Factor 11 is an active pharmaceutical agent.

“Active target region” or “target region” means a region to which one or more active antisense compounds is targeted. “Active antisense compounds” means antisense compounds that reduce target nucleic acid levels or protein levels.

“Administered concomitantly” refers to the co-administration of two agents in any manner in which the pharmacological effects of both are manifest in the patient at the same time. Concomitant administration does not require that both agents be administered in a single pharmaceutical composition, in the same dosage form, or by the same route of administration. The effects of both agents need not manifest themselves at the same time. The effects need only be overlapping for a period of time and need not be coextensive.

“Administering” means providing a pharmaceutical agent to an individual, and includes, but is not limited to administering by a medical professional and self-administering.

“Amelioration” refers to a lessening of at least one indicator, sign, or symptom of an associated disease, disorder, or condition. The severity of indicators may be determined by subjective or objective measures, which are known to those skilled in the art.

“Animal” refers to a human or non-human animal, including, but not limited to, mice, rats, rabbits, dogs, cats, pigs, and non-human primates, including, but not limited to, monkeys and chimpanzees.

“Antidote compound” refers to a compound capable of decreasing the intensity or duration of any antisense-mediated activity.

“Antidote oligonucleotide” means an antidote compound comprising an oligonucleotide that is complementary to and capable of hybridizing with an antisense compound.

“Antidote protein” means an antidote compound comprising a peptide.

“Antibody” refers to a molecule characterized by reacting specifically with an antigen in some way, where the antibody and the antigen are each defined in terms of the other. Antibody may refer to a complete antibody molecule or any fragment or region thereof, such as the heavy chain, the light chain, Fab region, and Fc region.

“Antisense activity” means any detectable or measurable activity attributable to the hybridization of an antisense compound to its target nucleic acid. In certain embodiments, antisense activity is a decrease in the amount or expression of a target nucleic acid or protein encoded by such target nucleic acid.

“Antisense compound” means an oligomeric compound that is capable of undergoing hybridization to a target nucleic acid through hydrogen bonding.

“Antisense inhibition” means reduction of target nucleic acid levels or target protein levels in the presence of an antisense compound complementary to a target nucleic acid compared to target nucleic acid levels or target protein levels in the absence of the antisense compound.

“Antisense oligonucleotide” means a single-stranded oligonucleotide having a nucleobase sequence that permits hybridization to a corresponding region or segment of a target nucleic acid.

“Bicyclic sugar” means a furosyl ring modified by the bridging of two non-geminal ring atoms. A bicyclic sugar is a modified sugar.

“Bicyclic nucleic acid” or “BNA” refers to a nucleoside or nucleotide wherein the furanose portion of the nucleoside or nucleotide includes a bridge connecting two carbon atoms on the furanose ring, thereby forming a bicyclic ring system.

“Cap structure” or “terminal cap moiety” means chemical modifications, which have been incorporated at either terminus of an antisense compound.

“Chemically distinct region” refers to a region of an antisense compound that is in some way chemically different than another region of the same antisense compound. For example, a region having 2′-O-methoxyethyl nucleotides is chemically distinct from a region having nucleotides without 2′-O-methoxyethyl modifications.

“Chimeric antisense compound” means an antisense compound that has at least two chemically distinct regions.

“Co-administration” means administration of two or more pharmaceutical agents to an individual. The two or more pharmaceutical agents may be in a single pharmaceutical composition, or may be in separate pharmaceutical compositions. Each of the two or more pharmaceutical agents may be administered through the same or different routes of administration. Co-administration encompasses parallel or sequential administration.

“Coagulation factor” means any of factors I, II, III, IV, V, VII, VIII, IX, X, XI, XII, XIII, or TAFI in the blood coagulation cascade. “Coagulation factor nucleic acid” means any nucleic acid encoding a coagulation factor. For example, in certain embodiments, a coagulation factor nucleic acid includes, without limitation, a DNA sequence encoding a coagulation factor (including genomic DNA comprising introns and exons), an RNA sequence transcribed from DNA encoding a coagulation factor, and an mRNA sequence encoding a coagulation factor. “Coagulation factor mRNA” means an mRNA encoding a coagulation factor protein.

“Complementarity” means the capacity for pairing between nucleobases of a first nucleic acid and a second nucleic acid.

“Contiguous nucleobases” means nucleobases immediately adjacent to each other.

“Diluent” means an ingredient in a composition that lacks pharmacological activity, but is pharmaceutically necessary or desirable. For example, the diluent in an injected composition may be a liquid, e.g. saline solution.

“Dose” means a specified quantity of a pharmaceutical agent provided in a single administration, or in a specified time period. In certain embodiments, a dose may be administered in one, two, or more boluses, tablets, or injections. For example, in certain embodiments where subcutaneous administration is desired, the desired dose requires a volume not easily accommodated by a single injection, therefore, two or more injections may be used to achieve the desired dose. In certain embodiments, the pharmaceutical agent is administered by infusion over an extended period of time or continuously. Doses may be stated as the amount of pharmaceutical agent per hour, day, week, or month.

“Effective amount” means the amount of active pharmaceutical agent sufficient to effectuate a desired physiological outcome in an individual in need of the agent. The effective amount may vary among individuals depending on the health and physical condition of the individual to be treated, the taxonomic group of the individuals to be treated, the formulation of the composition, assessment of the individual's medical condition, and other relevant factors.

“Factor 11 nucleic acid” or “Factor XI nucleic acid” or “F 11 nucleic acid” or “F XI nucleic acid” means any nucleic acid encoding Factor 11. For example, in certain embodiments, a Factor 11 nucleic acid includes a DNA sequence encoding Factor 11, an RNA sequence transcribed from DNA encoding Factor 11 (including genomic DNA comprising introns and exons), and an mRNA sequence encoding Factor 11. “Factor 11 mRNA” means an mRNA encoding a Factor 11 protein.

“Factor 11 specific inhibitor” refers to any agent capable of specifically inhibiting the expression of Factor 11 mRNA and/or Factor 11 protein at the molecular level. For example, Factor 11 specific inhibitors include nucleic acids (including antisense compounds), peptides, antibodies, small molecules, and other agents capable of inhibiting the expression of Factor 11 mRNA and/or Factor 11 protein. In certain embodiments, by specifically modulating Factor 11 mRNA expression and/or Factor 11 protein expression, Factor 11 specific inhibitors may affect other components of the coagulation cascade including downstream components. Similarly, in certain embodiments, Factor 11 specific inhibitors may affect other molecular processes in an animal.

“Factor 11 specific inhibitor antidote” means a compound capable of decreasing the effect of a Factor 11 specific inhibitor. In certain embodiments, a Factor 11 specific inhibitor antidote is selected from a Factor 11 peptide; a Factor 11 antidote oligonucleotide, including a Factor 11 antidote compound complementary to a Factor 11 antisense compound; and any compound or protein that affects the intrinsic or extrinsic coagulation pathway.

“Fully complementary” or “100% complementary” means each nucleobase of a first nucleic acid has a complementary nucleobase in a second nucleic acid. In certain embodiments, a first nucleic acid is an antisense compound and a target nucleic acid is a second nucleic acid.

“Gapmer” means a chimeric antisense compound in which an internal region having a plurality of nucleosides that support RNase H cleavage is positioned between external regions having one or more nucleosides, wherein the nucleosides comprising the internal region are chemically distinct from the nucleoside or nucleosides comprising the external regions. The internal region may be referred to as a “gap segment” and the external regions may be referred to as “wing segments.”

“Gap-widened” means a chimeric antisense compound having a gap segment of 12 or more contiguous 2′-deoxyribonucleosides positioned between and immediately adjacent to 5′ and 3′ wing segments having from one to six nucleosides.

“Hybridization” means the annealing of complementary nucleic acid molecules. In certain embodiments, complementary nucleic acid molecules include an antisense compound and a target nucleic acid.

“Identifying an animal at risk for thromboembolic complications” means identifying an animal having been diagnosed with a thromboembolic complication or identifying an animal predisposed to develop a thromboembolic complication. Individuals predisposed to develop a thromboembolic complication include those having one or more risk factors for thromboembolic complications including immobility, surgery (particularly orthopedic surgery), malignancy, pregnancy, older age, use of oral contraceptives, and inherited or acquired prothrombotic clotting disorders. Such identification may be accomplished by any method including evaluating an individual's medical history and standard clinical tests or assessments.

“Immediately adjacent” means there are no intervening elements between the immediately adjacent elements.

“Individual” means a human or non-human animal selected for treatment or therapy.

“Internucleoside linkage” refers to the chemical bond between nucleosides.

“Linked nucleosides” means adjacent nucleosides which are bonded together.

“Mismatch” or “non-complementary nucleobase” refers to the case when a nucleobase of a first nucleic acid is not capable of pairing with the corresponding nucleobase of a second or target nucleic acid.

“Modified internucleoside linkage” refers to a substitution or any change from a naturally occurring internucleoside bond (i.e. a phosphodiester internucleoside bond).

“Modified nucleobase” refers to any nucleobase other than adenine, cytosine, guanine, thymidine, or uracil. An “unmodified nucleobase” means the purine bases adenine (A) and guanine (G), and the pyrimidine bases thymine (T), cytosine (C), and uracil (U).

“Modified nucleotide” means a nucleotide having, independently, a modified sugar moiety, modified internucleoside linkage, or modified nucleobase. A “modified nucleoside” means a nucleoside having, independently, a modified sugar moiety or modified nucleobase.

“Modified oligonucleotide” means an oligonucleotide comprising a modified internucleoside linkage, a modified sugar, or a modified nucleobase.

“Modified sugar” refers to a substitution or change from a natural sugar.

“Motif” means the pattern of chemically distinct regions in an antisense compound.

“Naturally occurring internucleoside linkage” means a 3′ to 5′ phosphodiester linkage.

“Natural sugar moiety” means a sugar found in DNA (2′-H) or RNA (2′-OH).

“Nucleic acid” refers to molecules composed of monomeric nucleotides. A nucleic acid includes ribonucleic acids (RNA), deoxyribonucleic acids (DNA), single-stranded nucleic acids, double-stranded nucleic acids, small interfering ribonucleic acids (siRNA), and microRNAs (miRNA).

“Nucleobase” means a heterocyclic moiety capable of pairing with a base of another nucleic acid.

“Nucleobase sequence” means the order of contiguous nucleobases independent of any sugar, linkage, or nucleobase modification.

“Nucleoside” means a nucleobase linked to a sugar.

“Nucleoside mimetic” includes those structures used to replace the sugar or the sugar and the base and not necessarily the linkage at one or more positions of an oligomeric compound such as for example nucleoside mimetics having morpholino, cyclohexenyl, cyclohexyl, tetrahydropyranyl, bicyclo or tricyclo sugar mimetics e.g. non furanose sugar units. Nucleotide mimetic includes those structures used to replace the nucleoside and the linkage at one or more positions of an oligomeric compound such as for example peptide nucleic acids or morpholinos (morpholinos linked by —N(H)—C(═O)—O— or other non-phosphodiester linkage). Sugar surrogate overlaps with the slightly broader term nucleoside mimetic but is intended to indicate replacement of the sugar unit (furanose ring) only. The tetrahydropyranyl rings provided herein are illustrative of an example of a sugar surrogate wherein the furanose sugar group has been replaced with a tetrahydropyranyl ring system.

“Nucleotide” means a nucleoside having a phosphate group covalently linked to the sugar portion of the nucleoside.

“Oligomeric compound” or “oligomer” means a polymer of linked monomeric subunits which is capable of hybridizing to at least a region of a nucleic acid molecule.

“Oligonucleotide” means a polymer of linked nucleosides each of which can be modified or unmodified, independent one from another.

“Parenteral administration” means administration through injection or infusion. Parenteral administration includes subcutaneous administration, intravenous administration, intramuscular administration, intraarterial administration, intraperitoneal administration, or intracranial administration, e.g. intrathecal or intracerebroventricular administration.

“Peptide” means a molecule formed by linking at least two amino acids by amide bonds. Peptide refers to polypeptides and proteins.

“Pharmaceutical composition” means a mixture of substances suitable for administering to an individual. For example, a pharmaceutical composition may comprise one or more active pharmaceutical agents and a sterile aqueous solution.

“Pharmaceutically acceptable salts” means physiologically and pharmaceutically acceptable salts of antisense compounds, i.e., salts that retain the desired biological activity of the parent oligonucleotide and do not impart undesired toxicological effects thereto.

“Phosphorothioate linkage” means a linkage between nucleosides where the phosphodiester bond is modified by replacing one of the non-bridging oxygen atoms with a sulfur atom. A phosphorothioate linkage (P═S) is a modified internucleoside linkage.

“Portion” means a defined number of contiguous (i.e. linked) nucleobases of a nucleic acid. In certain embodiments, a portion is a defined number of contiguous nucleobases of a target nucleic acid. In certain embodiments, a portion is a defined number of contiguous nucleobases of an antisense compound.

“Prevent” refers to delaying or forestalling the onset or development of a disease, disorder, or condition for a period of time from minutes to indefinitely. Prevent also means reducing risk of developing a disease, disorder, or condition.

“Prodrug” means a therapeutic agent that is prepared in an inactive form that is converted to an active form within the body or cells thereof by the action of endogenous enzymes or other chemicals or conditions.

“Side effects” means physiological responses attributable to a treatment other than the desired effects. In certain embodiments, side effects include injection site reactions, liver function test abnormalities, renal function abnormalities, liver toxicity, renal toxicity, central nervous system abnormalities, myopathies, and malaise. For example, increased aminotransferase levels in serum may indicate liver toxicity or liver function abnormality. For example, increased bilirubin may indicate liver toxicity or liver function abnormality.

“Single-stranded oligonucleotide” means an oligonucleotide which is not hybridized to a complementary strand.

“Specifically hybridizable” refers to an antisense compound having a sufficient degree of complementarity between an antisense oligonucleotide and a target nucleic acid to induce a desired effect, while exhibiting minimal or no effects on non-target nucleic acids under conditions in which specific binding is desired, i.e. under physiological conditions in the case of in vivo assays and therapeutic treatments.

“Targeting” or “targeted” means the process of design and selection of an antisense compound that will specifically hybridize to a target nucleic acid and induce a desired effect.

“Target nucleic acid,” “target RNA,” and “target RNA transcript” all refer to a nucleic acid capable of being targeted by antisense compounds.

“Target segment” means the sequence of nucleotides of a target nucleic acid to which an antisense compound is targeted. “5′ target site” refers to the 5′-most nucleotide of a target segment. “3′ target site” refers to the 3′-most nucleotide of a target segment.

“Therapeutically effective amount” means an amount of a pharmaceutical agent that provides a therapeutic benefit to an individual.

“Thromboembolic complication” means any disease, disorder, or condition involving an embolism caused by a thrombus. Examples of such diseases, disorders, and conditions include the categories of thrombosis, embolism, and thromboembolism. In certain embodiments, such disease disorders, and conditions include deep vein thrombosis, pulmonary embolism, myocardial infarction, and stroke.

“Treat” refers to administering a pharmaceutical composition to effect an alteration or improvement of a disease, disorder, or condition.

“Unmodified nucleotide” means a nucleotide composed of naturally occurring nucleobases, sugar moieties, and internucleoside linkages. In certain embodiments, an unmodified nucleotide is an RNA nucleotide (i.e. f3-D-ribonucleosides) or a DNA nucleotide (i.e. β-D-deoxyribonucleoside).

Certain Embodiments

Embodiments of the present invention provide methods, compounds, and compositions for decreasing Factor 11 mRNA and protein expression.

Embodiments of the present invention provide methods, compounds, and compositions for the treatment, prevention, or amelioration of diseases, disorders, and conditions associated with Factor 11 in an individual in need thereof. Also contemplated are methods and compounds for the preparation of a medicament for the treatment, prevention, or amelioration of a disease, disorder, or condition associated with Factor 11. Factor 11 associated diseases, disorders, and conditions include thromboembolic complications such as thrombosis, embolism, thromboembolism, deep vein thrombosis, pulmonary embolism, myocardial infarction, and stroke.

Embodiments of the present invention provide a Factor 11 specific inhibitor for use in treating, preventing, or ameliorating a Factor 11 associated disease. In certain embodiments, Factor 11 specific inhibitors are nucleic acids (including antisense compounds), peptides, antibodies, small molecules, and other agents capable of inhibiting the expression of Factor 11 mRNA and/or Factor 11 protein.

In certain embodiments of the present invention, Factor 11 specific inhibitors are peptides or proteins, such as, but not limited to, alpha 1 protease inhibitors, antithrombin III, C1 inhibitors, and alpha 2 plasmin inhibitors as described in J Clin Invest 1982, 69:844-852; alpha 1 antitrypsin (alpha 1AT) as described in Thromb Res 1987, 48:145-151; Factor 11 peptide inhibitors as described in USPPN 2008/021998 and Blood 1998, 92:4198-206; MAP4-RGKWC as described in Thromb Res 2001, 104:451-465; beta 2 GPI as described in Proc Natl Acad Sci 2004, 101:3939-44; Lentinus proteinase inhibitor as described in Eur J Biochem 1999, 262:915-923; protease nexin-2/amyloid beta protein precursor Kunitz domain inhibitor (APPI) and antithrombin (AT) as described in J Biol Chem 2004, 279:29485-29492; and aprotinin as described in J Biol Chem 2005, 280:23523-30.

In certain embodiments of the present invention, Factor 11 specific inhibitors are antibodies, such as, but not limited to, Winston-Salem (IgG3 kappa) and Baltimore (IgG1 kappa) as described in Blood 1988, 72:1748-54; 5F4, 3C1, and 1F1 as described in J Biol Chem 1985, 260:10714-719; monoclonal antibodies as described in Throm Haemost 1990, 63:417-23; XI-5108 as described in J Thromb Haem 2006, 4:1496-1501; monoclonal antibodies 4-1 as described in Thromb Res 1986, 42:225-34; and abcixmab antibody as described in Example 19 of U.S. Pat. No. 6,566,140.

In certain embodiments of the present invention, Factor 11 specific inhibitors are small molecules, such as, but not limited to, diisopropyl fluorophosphates (DFP); the small molecule inhibitors as described in Examples 1-7 of USPPN 2004/0180855; and p-aminobenzamidine (pAB) as described in J Biol Chem 2005, 280:23523-30.

Embodiments of the present invention provide a Factor 11 specific inhibitor, as described herein, for use in treating, preventing, or ameliorating thromboembolic complications such as thrombosis, embolism, thromboembolism, deep vein thrombosis, pulmonary embolism, myocardial infarction, and stroke.

Embodiments of the present invention provide the use of Factor 11 specific inhibitors as described herein in the manufacture of a medicament for treating, ameliorating, or preventing a thromboembolic complication such as thrombosis, embolism, thromboembolism, deep vein thrombosis, pulmonary embolism, myocardial infarction, and stroke.

Embodiments of the present invention provide a Factor 11 specific inhibitor as described herein for use in treating, preventing, or ameliorating a thromboembolic complication as described herein by combination therapy with an additional agent or therapy as described herein. Agents or therapies can be co-administered or administered concomitantly.

Embodiments of the present invention provide the use of a Factor 11 specific inhibitor as described herein in the manufacture of a medicament for treating, preventing, or ameliorating a thromboembolic complication as described herein by combination therapy with an additional agent or therapy as described herein. Agents or therapies can be co-administered or administered concomitantly.

Embodiments of the present invention provide the use of a Factor 11 specific inhibitor as described herein in the manufacture of a medicament for treating, preventing, or ameliorating a thromboembolic complication as described herein in a patient who is subsequently administered an additional agent or therapy as described herein.

Embodiments of the present invention provide a kit for treating, preventing, or ameliorating a thromboembolic complication as described herein wherein the kit comprises:

(i) a Factor 11 specific inhibitor as described herein; and alternatively (ii) an additional agent or therapy as described herein.

A kit of the present invention may further include instructions for using the kit to treat, prevent, or ameliorate a thromboembolic complication as described herein by combination therapy as described herein.

Embodiments of the present invention provide antisense compounds targeted to a Factor 11 nucleic acid. In certain embodiments, the Factor 11 nucleic acid is any of the sequences set forth in GENBANK Accession No. NM_(—)000128.3 (incorporated herein as SEQ ID NO: 1), GENBANK Accession No. NT_(—)022792.17, truncated from 19598000 to 19624000, (incorporated herein as SEQ ID NO: 2), GENBANK Accession No. NM_(—)028066.1 (incorporated herein as SEQ ID NO: 6), exons 1-15 GENBANK Accession No. NW_(—)001118167.1 (incorporated herein as SEQ ID NO: 274).

In certain embodiments, the invention provides a compound comprising a modified oligonucleotide. In certain embodiments, the compound of the invention comprises a modified oligonucleotide consisting of 12 to 30 linked nucleosides.

In certain embodiments, the compound of the invention may comprise a modified oligonucleotide comprising a nucleobase sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% complementary to an equal length portion of SEQ ID NO: 1. In certain embodiments, the compound of the invention may comprise a modified oligonucleotide comprising a nucleobase sequence 100% complementary to an equal length portion of SEQ ID NO: 1.

In certain embodiments, the invention provides a compound comprising a modified oligonucleotide comprising a nucleobase sequence complementary to at least a portion of nucleobases 656 to 676 of SEQ ID NO: 1. Said modified oligonucleotide may comprise at least 8, at least 10, at least 12, at least 14, at least 16, at least 18 or 20 contiguous nucleobases complementary to an equal length portion of nucleobases 656 to 676 of SEQ ID NO: 1. Said modified oligonucleotide may comprise a nucleobase sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% complementary to an equal length portion of SEQ ID NO: 1. Said modified oligonucleotide may comprise a nucleobase sequence 100% complementary to an equal length portion of SEQ ID NO: 1. Said modified oligonucleotide may achieve at least 80% inhibition of human mRNA levels as determined using an RT-PCR assay method, optionally in HepG2 cells (e.g. as described in Example 3).

In certain embodiments, the invention provides a compound comprising a modified oligonucleotide comprising a nucleobase sequence complementary to at least a portion of nucleobases 665 to 687 of SEQ ID NO: 1. Said modified oligonucleotide may comprise at least 8, at least 10, at least 12, at least 14, at least 16, at least 18 or 20 contiguous nucleobases complementary to an equal length portion of nucleobases 665 to 687 of SEQ ID NO: 1. Said modified oligonucleotide may comprise a nucleobase sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% complementary to an equal length portion of SEQ ID NO: 1. Said modified oligonucleotide may comprise a nucleobase sequence 100% complementary to an equal length portion of SEQ ID NO: 1. Said modified oligonucleotide may achieve at least 50% inhibition of human mRNA levels as determined using an RT-PCR assay method, optionally in HepG2 cells (e.g. as described in Example 3).

In certain embodiments, the invention provides a compound comprising a modified oligonucleotide comprising a nucleobase sequence complementary to at least a portion of nucleobases 675 to 704 of SEQ ID NO: 1. Said modified oligonucleotide may comprise at least 8, at least 10, at least 12, at least 14, at least 16, at least 18 or 20 contiguous nucleobases complementary to an equal length portion of nucleobases 675 to 704 of SEQ ID NO: 1. Said modified oligonucleotide may comprise a nucleobase sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% complementary to an equal length portion of SEQ ID NO: 1. Said modified oligonucleotide may comprise a nucleobase sequence 100% complementary to an equal length portion of SEQ ID NO: 1. Said modified oligonucleotide may achieve at least 50% inhibition of human mRNA levels as determined using an RT-PCR assay method, optionally in HepG2 cells (e.g. as described in Example 3).

In certain embodiments, the invention provides a compound comprising a modified oligonucleotide comprising a nucleobase sequence complementary to at least a portion of nucleobases 677 to 704 of SEQ ID NO: 1. Said modified oligonucleotide may comprise at least 8, at least 10, at least 12, at least 14, at least 16, at least 18 or 20 contiguous nucleobases complementary to an equal length portion of nucleobases 677 to 704 of SEQ ID NO: 1. Said modified oligonucleotide may comprise a nucleobase sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% complementary to an equal length portion of SEQ ID NO: 1. Said modified oligonucleotide may comprise a nucleobase sequence 100% complementary to an equal length portion of SEQ ID NO: 1. Said modified oligonucleotide may achieve at least 60% inhibition of human mRNA levels as determined using an RT-PCR assay method, optionally in HepG2 cells (e.g. as described in Example 3).

In certain embodiments, the invention provides a compound comprising a modified oligonucleotide comprising a nucleobase sequence complementary to at least a portion of nucleobases 678 to 697 of SEQ ID NO: 1. Said modified oligonucleotide may comprise at least 8, at least 10, at least 12, at least 14, at least 16, at least 18 or 20 contiguous nucleobases complementary to an equal length portion of nucleobases 678 to 697 of SEQ ID NO: 1. Said modified oligonucleotide may comprise a nucleobase sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% complementary to an equal length portion of SEQ ID NO: 1. Said modified oligonucleotide may comprise a nucleobase sequence 100% complementary to an equal length portion of SEQ ID NO: 1. Said modified oligonucleotide may achieve at least 70% inhibition of human mRNA levels as determined using an RT-PCR assay method, optionally in HepG2 cells (e.g. as described in Example 3).

In certain embodiments, the invention provides a compound comprising a modified oligonucleotide comprising a nucleobase sequence complementary to at least a portion of nucleobases 680 to 703 of SEQ ID NO: 1. Said modified oligonucleotide may comprise at least 8, at least 10, at least 12, at least 14, at least 16, at least 18 or 20 contiguous nucleobases complementary to an equal length portion of nucleobases 680 to 703 of SEQ ID NO: 1. Said modified oligonucleotide may comprise a nucleobase sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% complementary to an equal length portion of SEQ ID NO: 1. Said modified oligonucleotide may comprise a nucleobase sequence 100% complementary to an equal length portion of SEQ ID NO: 1. Said modified oligonucleotide may achieve at least 80% inhibition of human mRNA levels as determined using an RT-PCR assay method, optionally in HepG2 cells (e.g. as described in Example 3 and Example 30).

In certain embodiments, the invention provides a compound comprising a modified oligonucleotide comprising a nucleobase sequence complementary to at least a portion of nucleobases 683 to 702 of SEQ ID NO: 1. Said modified oligonucleotide may comprise at least 8, at least 10, at least 12, at least 14, at least 16, at least 18 or 20 contiguous nucleobases complementary to an equal length portion of nucleobases 683 to 702 of SEQ ID NO: 1. Said modified oligonucleotide may comprise a nucleobase sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% complementary to an equal length portion of SEQ ID NO: 1. Said modified oligonucleotide may comprise a nucleobase sequence 100% complementary to an equal length portion of SEQ ID NO: 1. Said modified oligonucleotide may achieve at least 90% inhibition of human mRNA levels as determined using an RT-PCR assay method, optionally in HepG2 cells (e.g. as described in Example 3).

In certain embodiments, the invention provides a compound comprising a modified oligonucleotide comprising a nucleobase sequence complementary to at least a portion of nucleobases 738 to 759 of SEQ ID NO: 1. Said modified oligonucleotide may comprise at least 8, at least 10, at least 12, at least 14, at least 16, at least 18 or 20 contiguous nucleobases complementary to an equal length portion of nucleobases 738 to 759 of SEQ ID NO: 1. Said modified oligonucleotide may comprise a nucleobase sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% complementary to an equal length portion of SEQ ID NO: 1. Said modified oligonucleotide may comprise a nucleobase sequence 100% complementary to an equal length portion of SEQ ID NO: 1. Said modified oligonucleotide may achieve at least 80% inhibition of human mRNA levels as determined using an RT-PCR assay method, optionally in HepG2 cells (e.g. as described in Example 3 and Example 30).

In certain embodiments, the invention provides a compound comprising a modified oligonucleotide comprising a nucleobase sequence complementary to at least a portion of nucleobases 738 to 760 of SEQ ID NO: 1. Said modified oligonucleotide may comprise at least 8, at least 10, at least 12, at least 14, at least 16, at least 18 or 20 contiguous nucleobases complementary to an equal length portion of nucleobases 738 to 760 of SEQ ID NO: 1. Said modified oligonucleotide may comprise a nucleobase sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% complementary to an equal length portion of SEQ ID NO: 1. Said modified oligonucleotide may comprise a nucleobase sequence 100% complementary to an equal length portion of SEQ ID NO: 1. Said modified oligonucleotide may achieve at least 60% inhibition of human mRNA levels as determined using an RT-PCR assay method, optionally in HepG2 cells (e.g. as described in Example 3).

In certain embodiments, the invention provides a compound comprising a modified oligonucleotide comprising a nucleobase sequence complementary to at least a portion of nucleobases 738 to 762 of SEQ ID NO: 1. Said modified oligonucleotide may comprise at least 8, at least 10, at least 12, at least 14, at least 16, at least 18 or 20 contiguous nucleobases complementary to an equal length portion of nucleobases 738 to 762 of SEQ ID NO: 1. Said modified oligonucleotide may comprise a nucleobase sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% complementary to an equal length portion of SEQ ID NO: 1. Said modified oligonucleotide may comprise a nucleobase sequence 100% complementary to an equal length portion of SEQ ID NO: 1. Said modified oligonucleotide may achieve at least 45% inhibition of human mRNA levels as determined using an RT-PCR assay method, optionally in HepG2 cells (e.g. as described in Example 3).

In certain embodiments, the invention provides a compound comprising a modified oligonucleotide comprising a nucleobase sequence complementary to at least a portion of nucleobases 1018 to 1042 of SEQ ID NO: 1. Said modified oligonucleotide may comprise at least 8, at least 10, at least 12, at least 14, at least 16, at least 18 or 20 contiguous nucleobases complementary to an equal length portion of nucleobases 1018 to 1042 of SEQ ID NO: 1. Said modified oligonucleotide may comprise a nucleobase sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% complementary to an equal length portion of SEQ ID NO: 1. Said modified oligonucleotide may comprise a nucleobase sequence 100% complementary to an equal length portion of SEQ ID NO: 1. Said modified oligonucleotide may achieve at least 80% inhibition of human mRNA levels as determined using an RT-PCR assay method, optionally in HepG2 cells (e.g. as described in Example 3). In certain embodiments, the invention provides a compound comprising a modified oligonucleotide comprising a nucleobase sequence complementary to at least a portion of nucleobases 1062 to 1089 of SEQ ID NO: 1. Said modified oligonucleotide may comprise at least 8, at least 10, at least 12, at least 14, at least 16, at least 18 or 20 contiguous nucleobases complementary to an equal length portion of nucleobases 1062 to 1089 of SEQ ID NO: 1. Said modified oligonucleotide may comprise a nucleobase sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% complementary to an equal length portion of SEQ ID NO: 1. Said modified oligonucleotide may comprise a nucleobase sequence 100% complementary to an equal length portion of SEQ ID NO: 1. Said modified oligonucleotide may achieve at least 70% inhibition of human mRNA levels as determined using an RT-PCR assay method, optionally in HepG2 cells (e.g. as described in Example 3).

In certain embodiments, the invention provides a compound comprising a modified oligonucleotide comprising a nucleobase sequence complementary to at least a portion of nucleobases 1062 to 1090 of SEQ ID NO: 1. Said modified oligonucleotide may comprise at least 8, at least 10, at least 12, at least 14, at least 16, at least 18 or 20 contiguous nucleobases complementary to an equal length portion of nucleobases 1062 to 1090 of SEQ ID NO: 1. Said modified oligonucleotide may comprise a nucleobase sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% complementary to an equal length portion of SEQ ID NO: 1. Said modified oligonucleotide may comprise a nucleobase sequence 100% complementary to an equal length portion of SEQ ID NO: 1. Said modified oligonucleotide may achieve at least 60% inhibition of human mRNA levels as determined using an RT-PCR assay method, optionally in HepG2 cells (e.g. as described in Example 3).

In certain embodiments, the invention provides a compound comprising a modified oligonucleotide comprising a nucleobase sequence complementary to at least a portion of nucleobases 1062 to 1091 of SEQ ID NO: 1. Said modified oligonucleotide may comprise at least 8, at least 10, at least 12, at least 14, at least 16, at least 18 or 20 contiguous nucleobases complementary to an equal length portion of nucleobases 1062 to 1091 of SEQ ID NO: 1. Said modified oligonucleotide may comprise a nucleobase sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% complementary to an equal length portion of SEQ ID NO: 1. Said modified oligonucleotide may comprise a nucleobase sequence 100% complementary to an equal length portion of SEQ ID NO: 1. Said modified oligonucleotide may achieve at least 20% inhibition of human mRNA levels as determined using an RT-PCR assay method, optionally in HepG2 cells (e.g. as described in Example 3).

In certain embodiments, the invention provides a compound comprising a modified oligonucleotide comprising a nucleobase sequence complementary to at least a portion of nucleobases 1275 to 1301 of SEQ ID NO: 1. Said modified oligonucleotide may comprise at least 8, at least 10, at least 12, at least 14, at least 16, at least 18 or 20 contiguous nucleobases complementary to an equal length portion of nucleobases 1062 to 1091 of SEQ ID NO: 1. Said modified oligonucleotide may comprise a nucleobase sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% complementary to an equal length portion of SEQ ID NO: 1. Said modified oligonucleotide may comprise a nucleobase sequence 100% complementary to an equal length portion of SEQ ID NO: 1. Said modified oligonucleotide may achieve at least 80% inhibition of human mRNA levels as determined using an RT-PCR assay method, optionally in HepG2 cells (e.g. as described in Example 3).

In certain embodiments, the invention provides a compound comprising a modified oligonucleotide comprising a nucleobase sequence complementary to at least a portion of nucleobases 1276 to 1301 of SEQ ID NO: 1. Said modified oligonucleotide may comprise at least 8, at least 10, at least 12, at least 14, at least 16, at least 18 or 20 contiguous nucleobases complementary to an equal length portion of nucleobases 1062 to 1091 of SEQ ID NO: 1. Said modified oligonucleotide may comprise a nucleobase sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% complementary to an equal length portion of SEQ ID NO: 1. Said modified oligonucleotide may comprise a nucleobase sequence 100% complementary to an equal length portion of SEQ ID NO: 1. Said modified oligonucleotide may achieve at least 80% inhibition of human mRNA levels as determined using an RT-PCR assay method, optionally in HepG2 cells (e.g. as described in Example 30).

In certain embodiments, the invention provides a compound comprising a modified oligonucleotide comprising a nucleobase sequence complementary to at least a portion of nucleobases 1284 to 1308 of SEQ ID NO: 1. Said modified oligonucleotide may comprise at least 8, at least 10, at least 12, at least 14, at least 16, at least 18 or 20 contiguous nucleobases complementary to an equal length portion of nucleobases 1062 to 1091 of SEQ ID NO: 1. Said modified oligonucleotide may comprise a nucleobase sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% complementary to an equal length portion of SEQ ID NO: 1. Said modified oligonucleotide may comprise a nucleobase sequence 100% complementary to an equal length portion of SEQ ID NO: 1. Said modified oligonucleotide may achieve at least 80% inhibition of human mRNA levels as determined using an RT-PCR assay method, optionally in HepG2 cells (e.g. as described in Example 3).

In certain embodiments, the invention provides a compound comprising a modified oligonucleotide comprising a nucleobase sequence complementary to at least a portion of nucleobases 1291 to 1317 of SEQ ID NO: 1. Said modified oligonucleotide may comprise at least 8, at least 10, at least 12, at least 14, at least 16, at least 18 or 20 contiguous nucleobases complementary to an equal length portion of nucleobases 1062 to 1091 of SEQ ID NO: 1. Said modified oligonucleotide may comprise a nucleobase sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% complementary to an equal length portion of SEQ ID NO: 1. Said modified oligonucleotide may comprise a nucleobase sequence 100% complementary to an equal length portion of SEQ ID NO: 1. Said modified oligonucleotide may achieve at least 80% inhibition of human mRNA levels as determined using an RT-PCR assay method, optionally in HepG2 cells (e.g. as described in Example 3). In certain embodiments, the invention provides a compound comprising a modified oligonucleotide comprising a nucleobase sequence complementary to at least a portion of nucleobases 1275 to 1318 of SEQ ID NO: 1. Said modified oligonucleotide may comprise at least 8, at least 10, at least 12, at least 14, at least 16, at least 18 or 20 contiguous nucleobases complementary to an equal length portion of nucleobases 1275 to 1318 of SEQ ID NO: 1. Said modified oligonucleotide may comprise a nucleobase sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% complementary to an equal length portion of SEQ ID NO: 1. Said modified oligonucleotide may comprise a nucleobase sequence 100% complementary to an equal length portion of SEQ ID NO: 1. Said modified oligonucleotide may achieve at least 70% inhibition of human mRNA levels as determined using an RT-PCR assay method, optionally in HepG2 cells (e.g. as described in Example 3).

Embodiments of the present invention provide compounds comprising a modified oligonucleotide consisting of 12 to 30 linked nucleosides and having a nucleobase sequence comprising at least 8, at least 10, at least 12, at least 14, at least 16, at least 18, or 20 contiguous nucleobases of a nucleobase sequence selected from among the nucleobase sequences recited in SEQ ID NOs: 15 to 241.

Embodiments of the present invention provide compounds comprising a modified oligonucleotide consisting of 12 to 30 linked nucleosides and having a nucleobase sequence comprising at least 8, at least 10, at least 12, at least 14, at least 16, at least 18, or 20 contiguous nucleobases of a nucleobase sequence selected from among the nucleobase sequences recited in SEQ ID NOs: 15 to 269.

Embodiments of the present invention provide compounds comprising a modified oligonucleotide consisting of 12 to 30 linked nucleosides and having a nucleobase sequence comprising at least 8, at least 10, at least 12, at least 14, at least 16, at least 18, or 20 contiguous nucleobases of a nucleobase sequence selected from among the nucleobase sequences recited in SEQ ID NOs: 242 to 269.

In certain embodiments, the modified oligonucleotide comprises at least 8, at least 10, at least 12, at least 14, at least 16, or at least 18 nucleobases of a nucleobase sequence selected from ISIS Nos: 22, 31, 32, 34, 36 to 38, 40, 41, 43, 51 to 53, 55, 56, 59, 60, 64, 66, 71, 73, 75, 96, 98 to 103, 105 to 109, 113 to 117, 119, 124, 127, 129, 171, 172, 174, 176, 178, 179, 181 to 197, 199 to 211, and 213 to 232. In certain embodiments, the modified oligonucleotide comprises a nucleobase sequence selected from SEQ ID NOs: 22, 31, 32, 34, 36 to 38, 40, 41, 43, 51 to 53, 55, 56, 59, 60, 64, 66, 71, 73, 75, 96, 98 to 103, 105 to 109, 113 to 117, 119, 124, 127, 129, 171, 172, 174, 176, 178, 179, 181 to 197, 199 to 211, and 213 to 232. In certain embodiments, the modified oligonucleotide consists of a nucleobase sequence selected from SEQ ID NOs: 22, 31, 32, 34, 36 to 38, 40, 41, 43, 51 to 53, 55, 56, 59, 60, 64, 66, 71, 73, 75, 96, 98 to 103, 105 to 109, 113 to 117, 119, 124, 127, 129, 171, 172, 174, 176, 178, 179, 181 to 197, 199 to 211, and 213 to 232. Said modified oligonucleotide may achieve at least 70% inhibition of human mRNA levels as determined using an RT-PCR assay method, optionally in HepG2 cells (e.g. as described in Example 3).

In certain embodiments, the modified oligonucleotide comprises at least 8, at least 10, at least 12, at least 14, at least 16, or at least 18 nucleobases of a nucleobase sequence selected from ISIS Nos: 22, 31, 34, 37, 40, 43, 51 to 53, 60, 98, 100 to 102, 105 to 109, 114, 115, 119, 171, 174, 176, 179, 181, 186, 188 to 193, 195, 196, 199 to 210, and 213 to 232. In certain embodiments, the modified oligonucleotide comprises a nucleobase sequence selected from SEQ ID NOs: 22, 31, 34, 37, 40, 43, 51 to 53, 60, 98, 100 to 102, 105 to 109, 114, 115, 119, 171, 174, 176, 179, 181, 186, 188 to 193, 195, 196, 199 to 210, and 213 to 232. In certain embodiments, the modified oligonucleotide consists of a nucleobase sequence selected from SEQ ID NOs: 22, 31, 34, 37, 40, 43, 51 to 53, 60, 98, 100 to 102, 105 to 109, 114, 115, 119, 171, 174, 176, 179, 181, 186, 188 to 193, 195, 196, 199 to 210, and 213 to 232. Said modified oligonucleotide may achieve at least 80% inhibition of human mRNA levels as determined using an RT-PCR assay method, optionally in HepG2 cells (e.g. as described in Example 3).

In certain embodiments, the modified oligonucleotide comprises at least 8, at least 10, at least 12, at least 14, at least 16, or at least 18 nucleobases of a nucleobase sequence selected from ISIS Nos: 31, 37, 100, 105, 179, 190 to 193, 196, 202 to 207, 209, 210, 214 to 219, 221 to 224, 226, 227, 229, and 231. In certain embodiments, the modified oligonucleotide comprises a nucleobase sequence selected from SEQ ID NOs: 31, 37, 100, 105, 179, 190 to 193, 196, 202 to 207, 209, 210, 214 to 219, 221 to 224, 226, 227, 229, and 231. In certain embodiments, the modified oligonucleotide consists of a nucleobase sequence selected from SEQ ID NOs: 31, 37, 100, 105, 179, 190 to 193, 196, 202 to 207, 209, 210, 214 to 219, 221 to 224, 226, 227, 229, and 231. Said modified oligonucleotide may achieve at least 90% inhibition of human mRNA levels as determined using an RT-PCR assay method, optionally in HepG2 cells (e.g. as described in Example 3).

In certain embodiments, the modified oligonucleotide comprises at least 8, at least 10, at least 12, at least 14, at least 16, or at least 18 nucleobases of a nucleobase sequence selected from SEQ ID NOs: 34, 52, 53, 114, 115, 190, 213 to 232, 242 to 260, and 262 to 266. In certain embodiments, the modified oligonucleotide comprises a nucleobase sequence selected from SEQ ID NOs: 34, 52, 53, 114, 115, 190, 213 to 232, 242 to 260, and 262 to 266. In certain embodiments, the modified oligonucleotide consists of a nucleobase sequence selected from SEQ ID NOs: 34, 52, 53, 114, 115, 190, 213 to 232, 242 to 260, and 262 to 266. Said modified oligonucleotides may achieve at least 70% inhibition of human mRNA levels as determined using an RT-PCR assay method, optionally in HepG2 cells (e.g. as described in Example 30).

In certain embodiments, the modified oligonucleotide comprises at least 8, at least 10, at least 12, at least 14, at least 16, or at least 18 nucleobases of a nucleobase sequence selected from SEQ ID NOs: 34, 52, 53, 114, 115, 190, 213 to 216, 218 to 226, 243 to 246, 248, 249, 252 to 259, 264, and 265. In certain embodiments, the modified oligonucleotide comprises a nucleobase sequence selected from SEQ ID NOs: 34, 52, 53, 114, 115, 190, 213 to 216, 218 to 226, 243 to 246, 248, 249, 252 to 259, 264, and 265. In certain embodiments, the modified oligonucleotide consists of a nucleobase sequence selected from SEQ ID NOs: 34, 52, 53, 114, 115, 190, 213 to 216, 218 to 226, 243 to 246, 248, 249, 252 to 259, 264, and 265. Said modified oligonucleotides may achieve at least 80% inhibition of human mRNA levels as determined using an RT-PCR assay method, optionally in HepG2 cells (e.g. as described in Example 30).

In certain embodiments, the modified oligonucleotide comprises at least 8, at least 10, at least 12, at least 14, at least 16, or at least 18 nucleobases of a nucleobase sequence selected from SEQ ID NOs: 34, 190, 215, 222, 223, 226, 246, and 254. In certain embodiments, the modified oligonucleotide comprises a nucleobase sequence selected from SEQ ID NOs: 34, 190, 215, 222, 223, 226, 246, and 254. In certain embodiments, the modified oligonucleotide consists of a nucleobase sequence selected from SEQ ID NOs: 34, 190, 215, 222, 223, 226, 246, and 254. Said modified oligonucleotides may achieve at least 90% inhibition of human mRNA levels as determined using an RT-PCR assay method, optionally in HepG2 cells (e.g. as described in Example 30).

In certain embodiments, the compound consists of a single-stranded modified oligonucleotide.

In certain embodiments, the modified oligonucleotide consists of 20 linked nucleosides. In certain embodiments, the nucleobase sequence of the modified oligonucleotide is 100% complementary to a nucleobase sequence of SEQ ID NO: 1 or SEQ ID NO: 2 or SEQ ID NO: 6 or SEQ ID NO: 274.

In certain embodiments, the compound has at least one modified internucleoside linkage. In certain embodiments, the internucleoside linkage is a phosphorothioate internucleoside linkage.

In certain embodiments, the compound has at least one nucleoside comprising a modified sugar. In certain embodiments, the at least one modified sugar is a bicyclic sugar. In certain embodiments, the at least one modified sugar comprises a 2′-O-methoxyethyl.

Embodiments of the present invention provide compounds comprising a modified oligonucleotide consisting of 12 to 30 linked nucleosides and having a nucleobase sequence comprising at least 8, at least 10, at least 12, at least 14, at least 16, at least 18, or 20 contiguous nucleobases of a nucleobase sequence selected from among the nucleobase sequences recited in SEQ ID NOs: 15 to 241, SEQ ID NOs: 15 to 269, or SEQ ID NOs: 242 to 269, wherein at least one nucleoside comprises a modified sugar.

In certain embodiments, said at least one at least one modified sugar is a bicyclic sugar. In certain embodiments, said at least one bicyclic sugar comprises a 4′-(CH₂)_(n)—O-2′ bridge, wherein n is 1 or 2.

In certain embodiments, said at least one bicyclic sugar comprises a 4′-CH(CH₃)—O-2′ bridge.

In certain embodiments, said at least one modified sugar comprises a 2′-O-methoxyethyl group.

Embodiments of the present invention provide compounds comprising a modified oligonucleotide consisting of 12 to 30 linked nucleosides and having a nucleobase sequence comprising at least 8, at least 10, at least 12, at least 14, at least 16, at least 18, or 20 contiguous nucleobases of a nucleobase sequence selected from among the nucleobase sequences recited in SEQ ID NOs: 15 to 241, SEQ ID NOs: 15 to 269, or SEQ ID NOs: 242 to 269, comprising at least one tetrahydropyran modified nucleoside wherein a tetrahydropyran ring replaces the furanose ring.

In certain embodiments, said at least one tetrahydropyran modified nucleoside has the structure:

In certain embodiments, the compound has at least one nucleoside comprising a modified nucleobase. In certain embodiments, the modified nucleobase is a 5-methylcytosine.

In certain embodiments, the modified oligonucleotide of the compound comprises:

(i) a gap segment consisting of linked deoxynucleosides; (ii) a 5′ wing segment consisting of linked nucleosides; (iii) a 3′ wing segment consisting of linked nucleosides, wherein the gap segment is positioned immediately adjacent to and between the 5′ wing segment and the 3′ wing segment and wherein each nucleoside of each wing segment comprises a modified sugar. In some such embodiments, each cytosine in the modified oligonucleotide is a 5-methylcytosine.

In certain embodiments, the modified oligonucleotide of the compound comprises:

(i) a gap segment consisting of ten linked deoxynucleosides; (ii) a 5′ wing segment consisting of five linked nucleosides; (iii) a 3′ wing segment consisting of five linked nucleosides, wherein the gap segment is positioned immediately adjacent to and between the 5′ wing segment and the 3′ wing segment, wherein each nucleoside of each wing segment comprises a 2′-O-methoxyethyl sugar; and wherein each internucleoside linkage is a phosphorothioate linkage. In some such embodiments, each cytosine in the modified oligonucleotide is a 5-methylcytosine.

In certain embodiments, the modified oligonucleotide of the compound comprises:

(i) a gap segment consisting of fourteen linked deoxynucleosides; (ii) a 5′ wing segment consisting of three linked nucleosides; (iii) a 3′ wing segment consisting of three linked nucleosides, wherein the gap segment is positioned immediately adjacent to and between the 5′ wing segment and the 3′ wing segment, wherein each nucleoside of each wing segment comprises a 2′-O-methoxyethyl sugar; and wherein each internucleoside linkage is a phosphorothioate linkage. In some such embodiments, each cytosine in the modified oligonucleotide is a 5-methylcytosine.

In certain embodiments, the modified oligonucleotide of the compound comprises:

(i) a gap segment consisting of thirteen linked deoxynucleosides; (ii) a 5′ wing segment consisting of two linked nucleosides; (iii) a 3′ wing segment consisting of five linked nucleosides, wherein the gap segment is positioned immediately adjacent to and between the 5′ wing segment and the 3′ wing segment, wherein each nucleoside of each wing segment comprises a 2′-O-methoxyethyl sugar; and wherein each internucleoside linkage is a phosphorothioate linkage. In some such embodiments, each cytosine in the modified oligonucleotide is a 5-methylcytosine.

Embodiments of the present invention provide a composition comprising a modified oligonucleotide consisting of 12 to 30 linked nucleosides and having a nucleobase sequence comprising at least 12 contiguous nucleobases of a nucleobase sequence selected from among the nucleobase sequences recited in SEQ ID NOs: 15 to 241 or a salt thereof and a pharmaceutically acceptable carrier or diluent.

Embodiments of the present invention provide a composition comprising a modified oligonucleotide consisting of 12 to 30 linked nucleosides and having a nucleobase sequence comprising at least 12 contiguous nucleobases of a nucleobase sequence selected from among the nucleobase sequences recited in SEQ ID NOs: 15 to 269 or a salt thereof and a pharmaceutically acceptable carrier or diluent.

Embodiments of the present invention provide a composition comprising a modified oligonucleotide consisting of 12 to 30 linked nucleosides and having a nucleobase sequence comprising at least 12 contiguous nucleobases of a nucleobase sequence selected from among the nucleobase sequences recited in SEQ ID NOs: 241 to 269 or a salt thereof and a pharmaceutically acceptable carrier or diluent.

Embodiments of the present invention provide methods comprising administering to an animal a compound comprising a modified oligonucleotide consisting of 12 to 30 linked nucleosides and having a nucleobase sequence comprising at least 8 contiguous nucleobases of a nucleobase sequence selected from among the nucleobase sequences recited in SEQ ID NOs: 15 to 241.

Embodiments of the present invention provide methods comprising administering to an animal a compound comprising a modified oligonucleotide consisting of 12 to 30 linked nucleosides and having a nucleobase sequence comprising at least 8 contiguous nucleobases of a nucleobase sequence selected from among the nucleobase sequences recited in SEQ ID NOs: 15 to 269.

Embodiments of the present invention provide methods comprising administering to an animal a compound comprising a modified oligonucleotide consisting of 12 to 30 linked nucleosides and having a nucleobase sequence comprising at least 8 contiguous nucleobases of a nucleobase sequence selected from among the nucleobase sequences recited in SEQ ID NOs: 241 to 269.

In certain embodiments, the animal is a human.

In certain embodiments, the administering prevents deep vein thrombosis or pulmonary embolism.

In certain embodiments, the compound is co-administered with any of the group selected from aspirin, clopidogrel, dipyridamole, heparin, lepirudin, ticlopidine, warfarin, apixaban, rivaroxaban, and LOVENOX.

In certain embodiments, the compound is co-administered with any Factor Xa inhibitor.

In certain embodiment, the Factor Xa inhibitor is any of Rivaroxaban, LY517717, YM150, apixaban, PRT054021, and DU-176b.

In certain embodiments, the compound is administered concomitantly with any of the group selected from aspirin, clopidogrel, dipyridamole, heparin, lepirudin, ticlopidine, warfarin, apixaban, rivaroxaban, and LOVENOX are administered concomitantly.

In certain embodiments, the administering is parenteral administration. In certain embodiments, the parenteral administration is any of subcutaneous or intravenous administration.

Embodiments of the present invention provide methods comprising identifying an animal at risk for developing thromboembolic complications and administering to the at risk animal a therapeutically effective amount of a compound comprising a modified oligonucleotide consisting of 12 to 30 linked nucleosides, wherein the modified oligonucleotide is complementary to a Factor 11 nucleic acid.

In certain embodiments, the thromboembolic complication is deep vein thrombosis, pulmonary embolism, or a combination thereof.

Embodiments of the present invention provide methods comprising identifying an animal having a clotting disorder by administering to the animal a therapeutically effective amount of a compound comprising a modified oligonucleotide consisting of 12 to 30 linked nucleosides, wherein the modified oligonucleotide is complementary to a Factor 11 nucleic acid.

In certain embodiments, the compound is co-administered with any of the group selected from aspirin, clopidogrel, dipyridamole, heparin, lepirudin, ticlopidine, warfarin, apixaban, rivaroxaban, and LOVENOX.

In certain embodiments, the compound is administered concomitantly with any of the group selected from aspirin, clopidogrel, dipyridamole, heparin, lepirudin, ticlopidine, warfarin, apixaban, rivaroxaban, and LOVENOX are administered concomitantly.

Embodiments of the present invention provide methods comprising reducing the risk for thromboembolic complications in an animal by administering to the animal a therapeutically effective amount of a compound comprising a modified oligonucleotide consisting of 12 to 30 linked nucleosides, wherein the modified oligonucleotide is complementary to a Factor 11 nucleic acid.

Embodiments of the present invention provide methods comprising treating a clotting disorder in an animal by administering to the animal a therapeutically effective amount of a compound comprising a modified oligonucleotide consisting of 12 to 30 linked nucleosides, wherein the modified oligonucleotide is complementary to a Factor 11 nucleic acid.

Embodiments of the present invention provide methods comprising inhibiting Factor 11 expression in an animal by administering to the animal a therapeutically effective amount of a compound comprising a modified oligonucleotide consisting of 12 to 30 linked nucleosides, wherein the modified oligonucleotide is complementary to a Factor 11 nucleic acid.

In certain embodiments, the Factor 11 inhibition in the animal is reversed by administering an antidote to the modified oligonucleotide.

In certain embodiments, the antidote is an oligonucleotide complementary to the modified oligonucleotide.

Antisense Compounds

Oligomeric compounds include, but are not limited to, oligonucleotides, oligonucleosides, oligonucleotide analogs, oligonucleotide mimetics, antisense compounds, antisense oligonucleotides, and siRNAs. An oligomeric compound may be “antisense” to a target nucleic acid, meaning that is capable of undergoing hybridization to a target nucleic acid through hydrogen bonding.

In certain embodiments, an antisense compound has a nucleobase sequence that, when written in the 5′ to 3′ direction, comprises the reverse complement of the target segment of a target nucleic acid to which it is targeted. In certain such embodiments, an antisense oligonucleotide has a nucleobase sequence that, when written in the 5′ to 3′ direction, comprises the reverse complement of the target segment of a target nucleic acid to which it is targeted.

In certain embodiments, an antisense compound targeted to a Factor 11 nucleic acid is 12 to 30 subunits in length. In other words, such antisense compounds are from 12 to 30 linked subunits. In other embodiments, the antisense compound is 8 to 80, 12 to 50, 15 to 30, 18 to 24, 19 to 22, or linked subunits. In certain such embodiments, the antisense compounds are 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, or 80 linked subunits in length, or a range defined by any two of the above values. In some embodiments the antisense compound is an antisense oligonucleotide, and the linked subunits are nucleotides.

In certain embodiments anitsense oligonucleotides targeted to a Factor 11 nucleic acid may be shortened or truncated. For example, a single subunit may be deleted from the 5′ end (5′ truncation), or alternatively from the 3′ end (3′ truncation). A shortened or truncated antisense compound targeted to a Factor 11 nucleic acid may have two subunits deleted from the 5′ end, or alternatively may have two subunits deleted from the 3′ end, of the antisense compound. Alternatively, the deleted nucleosides may be dispersed throughout the antisense compound, for example, in an antisense compound having one nucleoside deleted from the 5′ end and one nucleoside deleted from the 3′ end.

When a single additional subunit is present in a lengthened antisense compound, the additional subunit may be located at the 5′ or 3′ end of the antisense compound. When two or more additional subunits are present, the added subunits may be adjacent to each other, for example, in an antisense compound having two subunits added to the 5′ end (5′ addition), or alternatively to the 3′ end (3′ addition), of the antisense compound. Alternatively, the added subunits may be dispersed throughout the antisense compound, for example, in an antisense compound having one subunit added to the 5′ end and one subunit added to the 3′ end.

It is possible to increase or decrease the length of an antisense compound, such as an antisense oligonucleotide, and/or introduce mismatch bases without eliminating activity. For example, in Woolf et al. (Proc. Natl. Acad. Sci. USA 89:7305-7309, 1992), a series of antisense oligonucleotides 13-25 nucleobases in length were tested for their ability to induce cleavage of a target RNA in an oocyte injection model. Antisense oligonucleotides 25 nucleobases in length with 8 or 11 mismatch bases near the ends of the antisense oligonucleotides were able to direct specific cleavage of the target mRNA, albeit to a lesser extent than the antisense oligonucleotides that contained no mismatches. Similarly, target specific cleavage was achieved using 13 nucleobase antisense oligonucleotides, including those with 1 or 3 mismatches.

Gautschi et al (J. Natl. Cancer Inst. 93:463-471, March 2001) demonstrated the ability of an oligonucleotide having 100% complementarity to the bcl-2 mRNA and having 3 mismatches to the bcl-xL mRNA to reduce the expression of both bcl-2 and bcl-xL in vitro and in vivo. Furthermore, this oligonucleotide demonstrated potent anti-tumor activity in vivo.

Maher and Dolnick (Nuc. Acid. Res. 16:3341-3358, 1988) tested a series of tandem 14 nucleobase antisense oligonucleotides, and a 28 and 42 nucleobase antisense oligonucleotides comprised of the sequence of two or three of the tandem antisense oligonucleotides, respectively, for their ability to arrest translation of human DHFR in a rabbit reticulocyte assay. Each of the three 14 nucleobase antisense oligonucleotides alone was able to inhibit translation, albeit at a more modest level than the 28 or 42 nucleobase antisense oligonucleotides.

Antisense Compound Motifs

In certain embodiments, antisense compounds targeted to a Factor 11 nucleic acid have chemically modified subunits arranged in patterns, or motifs, to confer to the antisense compounds properties such as enhanced the inhibitory activity, increased binding affinity for a target nucleic acid, or resistance to degradation by in vivo nucleases.

Chimeric antisense compounds typically contain at least one region modified so as to confer increased resistance to nuclease degradation, increased cellular uptake, increased binding affinity for the target nucleic acid, and/or increased inhibitory activity. A second region of a chimeric antisense compound may optionally serve as a substrate for the cellular endonuclease RNase H, which cleaves the RNA strand of an RNA:DNA duplex.

Antisense compounds having a gapmer motif are considered chimeric antisense compounds. In a gapmer an internal region having a plurality of nucleotides that supports RNaseH cleavage is positioned between external regions having a plurality of nucleotides that are chemically distinct from the nucleosides of the internal region. In the case of an antisense oligonucleotide having a gapmer motif, the gap segment generally serves as the substrate for endonuclease cleavage, while the wing segments comprise modified nucleosides. In certain embodiments, the regions of a gapmer are differentiated by the types of sugar moieties comprising each distinct region. The types of sugar moieties that are used to differentiate the regions of a gapmer may in some embodiments include β3-D-ribonucleosides, β-D-deoxyribonucleosides, 2′-modified nucleosides (such 2′-modified nucleosides may include 2′-MOE, and 2′-O—CH₃, among others), and bicyclic sugar modified nucleosides (such bicyclic sugar modified nucleosides may include those having a 4′-(CH₂)_(n)—O-2′ bridge, where n=1 or n=2). Preferably, each distinct region comprises uniform sugar moieties. The wing-gap-wing motif is frequently described as “X-Y-Z”, where “X” represents the length of the 5′ wing region, “Y” represents the length of the gap region, and “Z” represents the length of the 3′ wing region. As used herein, a gapmer described as “X-Y-Z” has a configuration such that the gap segment is positioned immediately adjacent each of the 5′ wing segment and the 3′ wing segment. Thus, no intervening nucleotides exist between the 5′ wing segment and gap segment, or the gap segment and the 3′ wing segment. Any of the antisense compounds described herein can have a gapmer motif. In some embodiments, X and Z are the same, in other embodiments they are different. In a preferred embodiment, Y is between 8 and 15 nucleotides. X, Y or Z can be any of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30 or more nucleotides. Thus, gapmers of the present invention include, but are not limited to, for example 5-10-5, 4-8-4, 4-12-3, 4-12-4, 3-14-3, 2-13-5, 2-16-2, 1-18-1, 3-10-3, 2-10-2, 1-10-1, 2-8-2, 5-8-5, or 6-8-6.

In certain embodiments, the antisense compound has a “wingmer” motif, having a wing-gap or gap-wing configuration, i.e. an X-Y or Y-Z configuration as described above for the gapmer configuration. Thus, wingmer configurations of the present invention include, but are not limited to, for example 5-10, 8-4, 4-12, 12-4, 3-14, 16-2, 18-1, 10-3, 2-10, 1-10, 8-2, 2-13, 5-13, 5-8, or 6-8.

In certain embodiments, antisense compounds targeted to a Factor 11 nucleic acid possess a 5-10-5 gapmer motif.

In certain embodiments, antisense compounds targeted to a Factor 11 nucleic acid possess a 3-14-3 gapmer motif.

In certain embodiments, antisense compounds targeted to a Factor 11 nucleic acid possess a 2-13-5 gapmer motif.

In certain embodiments, antisense compounds targeted to a Factor 11 nucleic acid possess a 5-8-5 gapmer motif.

In certain embodiments, antisense compounds targeted to a Factor 11 nucleic acid possess a 6-8-6 gapmer motif.

In certain embodiments, an antisense compound targeted to a Factor 11 nucleic acid has a gap-widened motif.

In certain embodiments, a gap-widened antisense oligonucleotide targeted to a Factor 11 nucleic acid has a gap segment of fourteen 2′-deoxyribonucleotides positioned immediately adjacent to and between wing segments of three chemically modified nucleosides. In certain embodiments, the chemical modification comprises a 2′-sugar modification. In another embodiment, the chemical modification comprises a 2′-MOE sugar modification.

In certain embodiments, a gap-widened antisense oligonucleotide targeted to a Factor 11 nucleic acid has a gap segment of thirteen 2′-deoxyribonucleotides positioned immediately adjacent to and between a 5′ wing segment of two chemically modified nucleosides and a 3′ wing segment of five chemically modified nucleosides. In certain embodiments, the chemical modification comprises a 2′-sugar modification. In another embodiment, the chemical modification comprises a 2′-MOE sugar modification.

Target Nucleic Acids, Target Regions and Nucleotide Sequences

Nucleotide sequences that encode Factor 11 include, without limitation, the following: GENBANK Accession No. NM_(—)000128.3, first deposited with GENBANK on Mar. 24, 1999 incorporated herein as SEQ ID NO: 1; NT_(—)022792.17, truncated from 19598000 to 19624000, first deposited with GENBANK on Nov. 29, 2000, and incorporated herein as SEQ ID NO: 2; GENBANK Accession No. NM_(—)028066.1, first deposited with GENBANK on Jun. 2, 2002, incorporated herein as SEQ ID NO: 6; and exons 1-15 GENBANK Accession No. NW_(—)001118167.1, first deposited with GENBANK on Mar. 28, 2006, incorporated herein as SEQ ID NO: 274.

It is understood that the sequence set forth in each SEQ ID NO in the Examples contained herein is independent of any modification to a sugar moiety, an internucleoside linkage, or a nucleobase. As such, antisense compounds defined by a SEQ ID NO may comprise, independently, one or more modifications to a sugar moiety, an internucleoside linkage, or a nucleobase. Antisense compounds described by Isis Number (Isis No) indicate a combination of nucleobase sequence and motif.

In certain embodiments, a target region is a structurally defined region of the target nucleic acid. For example, a target region may encompass a 3′ UTR, a 5′ UTR, an exon, an intron, an exon/intron junction, a coding region, a translation initiation region, translation termination region, or other defined nucleic acid region. The structurally defined regions for Factor 11 can be obtained by accession number from sequence databases such as NCBI and such information is incorporated herein by reference. In certain embodiments, a target region may encompass the sequence from a 5′ target site of one target segment within the target region to a 3′ target site of another target segment within the same target region.

Targeting includes determination of at least one target segment to which an antisense compound hybridizes, such that a desired effect occurs. In certain embodiments, the desired effect is a reduction in mRNA target nucleic acid levels. In certain embodiments, the desired effect is reduction of levels of protein encoded by the target nucleic acid or a phenotypic change associated with the target nucleic acid.

A target region may contain one or more target segments. Multiple target segments within a target region may be overlapping. Alternatively, they may be non-overlapping. In certain embodiments, target segments within a target region are separated by no more than about 300 nucleotides. In certain emodiments, target segments within a target region are separated by a number of nucleotides that is, is about, is no more than, is no more than about, 250, 200, 150, 100, 90, 80, 70, 60, 50, 40, 30, 20, or 10 nucleotides on the target nucleic acid, or is a range defined by any two of the preceeding values. In certain embodiments, target segments within a target region are separated by no more than, or no more than about, 5 nucleotides on the target nucleic acid. In certain embodiments, target segments are contiguous. Contemplated are target regions defined by a range having a starting nucleic acid that is any of the 5′ target sites or 3′ target sites listed herein.

Suitable target segments may be found within a 5′ UTR, a coding region, a 3′ UTR, an intron, an exon, or an exon/intron junction. Target segments containing a start codon or a stop codon are also suitable target segments. A suitable target segment may specifically exclude a certain structurally defined region such as the start codon or stop codon.

The determination of suitable target segments may include a comparison of the sequence of a target nucleic acid to other sequences throughout the genome. For example, the BLAST algorithm may be used to identify regions of similarity amongst different nucleic acids. This comparison can prevent the selection of antisense compound sequences that may hybridize in a non-specific manner to sequences other than a selected target nucleic acid (i.e., non-target or off-target sequences).

There may be variation in activity (e.g., as defined by percent reduction of target nucleic acid levels) of the antisense compounds within an active target region. In certain embodiments, reductions in Factor 11 mRNA levels are indicative of inhibition of Factor 11 expression. Reductions in levels of a Factor 11 protein are also indicative of inhibition of target mRNA expression. Further, phenotypic changes are indicative of inhibition of Factor 11 expression. For example, a prolonged aPTT time can be indicative of inhibition of Factor 11 expression. In another example, prolonged aPTT time in conjunction with a normal PT time can be indicative of inhibition of Factor 11 expression. In another example, a decreased quantity of Platelet Factor 4 (PF-4) can be indicative of inhibition of Factor 11 expression. In another example, reduced formation of thrombus or increased time for thrombus formation can be indicative of inhibition of Factor 11 expression.

Hybridization

In some embodiments, hybridization occurs between an antisense compound disclosed herein and a Factor 11 nucleic acid. The most common mechanism of hybridization involves hydrogen bonding (e.g., Watson-Crick, Hoogsteen or reversed Hoogsteen hydrogen bonding) between complementary nucleobases of the nucleic acid molecules.

Hybridization can occur under varying conditions. Stringent conditions are sequence-dependent and are determined by the nature and composition of the nucleic acid molecules to be hybridized.

Methods of determining whether a sequence is specifically hybridizable to a target nucleic acid are well known in the art. In certain embodiments, the antisense compounds provided herein are specifically hybridizable with a Factor 11 nucleic acid.

Complementarily

An antisense compound and a target nucleic acid are complementary to each other when a sufficient number of nucleobases of the antisense compound can hydrogen bond with the corresponding nucleobases of the target nucleic acid, such that a desired effect will occur (e.g., antisense inhibition of a target nucleic acid, such as a Factor 11 nucleic acid).

Non-complementary nucleobases between an antisense compound and a Factor 11 nucleic acid may be tolerated provided that the antisense compound remains able to specifically hybridize to a target nucleic acid. Moreover, an antisense compound may hybridize over one or more segments of a Factor 11 nucleic acid such that intervening or adjacent segments are not involved in the hybridization event (e.g., a loop structure, mismatch or hairpin structure).

In certain embodiments, the antisense compounds provided herein, or a specified portion thereof, are, or are at least, 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% complementary to a Factor 11 nucleic acid, a target region, target segment, or specified portion thereof. Percent complementarity of an antisense compound with a target nucleic acid can be determined using routine methods.

For example, an antisense compound in which 18 of 20 nucleobases of the antisense compound are complementary to a target region, and would therefore specifically hybridize, would represent 90 percent complementarity. In this example, the remaining noncomplementary nucleobases may be clustered or interspersed with complementary nucleobases and need not be contiguous to each other or to complementary nucleobases. As such, an antisense compound which is 18 nucleobases in length having 4 (four) noncomplementary nucleobases which are flanked by two regions of complete complementarity with the target nucleic acid would have 77.8% overall complementarity with the target nucleic acid and would thus fall within the scope of the present invention. Percent complementarity of an antisense compound with a region of a target nucleic acid can be determined routinely using BLAST programs (basic local alignment search tools) and PowerBLAST programs known in the art (Altschul et al., J. Mol. Biol., 1990, 215, 403 410; Zhang and Madden, Genome Res., 1997, 7, 649 656). Percent homology, sequence identity or complementarity, can be determined by, for example, the Gap program (Wisconsin Sequence Analysis Package, Version 8 for Unix, Genetics Computer Group, University Research Park, Madison Wis.), using default settings, which uses the algorithm of Smith and Waterman (Adv. Appl. Math., 1981, 2, 482 489).

In certain embodiments, the antisense compounds provided herein, or specified portions thereof, are fully complementary (i.e. 100% complementary) to a target nucleic acid, or specified portion thereof. For example, antisense compound may be fully complementary to a Factor 11 nucleic acid, or a target region, or a target segment or target sequence thereof. As used herein, “fully complementary” means each nucleobase of an antisense compound is capable of precise base pairing with the corresponding nucleobases of a target nucleic acid. For example, a 20 nucleobase antisense compound is fully complementary to a target sequence that is 400 nucleobases long, so long as there is a corresponding 20 nucleobase portion of the target nucleic acid that is fully complementary to the antisense compound. Fully complementary can also be used in reference to a specified portion of the first and/or the second nucleic acid. For example, a 20 nucleobase portion of a 30 nucleobase antisense compound can be “fully complementary” to a target sequence that is 400 nucleobases long. The 20 nucleobase portion of the 30 nucleobase oligonucleotide is fully complementary to the target sequence if the target sequence has a corresponding 20 nucleobase portion wherein each nucleobase is complementary to the 20 nucleobase portion of the antisense compound. At the same time, the entire 30 nucleobase antisense compound may or may not be fully complementary to the target sequence, depending on whether the remaining 10 nucleobases of the antisense compound are also complementary to the target sequence.

The location of a non-complementary nucleobase may be at the 5′ end or 3′ end of the antisense compound. Alternatively, the non-complementary nucleobase or nucleobases may be at an internal position of the antisense compound. When two or more non-complementary nucleobases are present, they may be contiguous (i.e. linked) or non-contiguous. In one embodiment, a non-complementary nucleobase is located in the wing segment of a gapmer antisense oligonucleotide.

In certain embodiments, antisense compounds that are, or are up to 12, 13, 14, 15, 16, 17, 18, 19, or 20 nucleobases in length comprise no more than 4, no more than 3, no more than 2, or no more than 1 non-complementary nucleobase(s) relative to a target nucleic acid, such as a Factor 11 nucleic acid, or specified portion thereof.

In certain embodiments, antisense compounds that are, or are up to 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleobases in length comprise no more than 6, no more than 5, no more than 4, no more than 3, no more than 2, or no more than 1 non-complementary nucleobase(s) relative to a target nucleic acid, such as a Factor 11 nucleic acid, or specified portion thereof.

The antisense compounds provided herein also include those which are complementary to a portion of a target nucleic acid. As used herein, “portion” refers to a defined number of contiguous (i.e. linked) nucleobases within a region or segment of a target nucleic acid. A “portion” can also refer to a defined number of contiguous nucleobases of an antisense compound. In certain embodiments, the antisense compounds, are complementary to at least an 8 nucleobase portion of a target segment. In certain embodiments, the antisense compounds are complementary to at least a 12 nucleobase portion of a target segment. In certain embodiments, the antisense compounds are complementary to at least a 15 nucleobase portion of a target segment. Also contemplated are antisense compounds that are complementary to at least a 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more nucleobase portion of a target segment, or a range defined by any two of these values.

Identity

The antisense compounds provided herein may also have a defined percent identity to a particular nucleotide sequence, SEQ ID NO, or compound represented by a specific Isis number, or portion thereof. As used herein, an antisense compound is identical to the sequence disclosed herein if it has the same nucleobase pairing ability. For example, a RNA which contains uracil in place of thymidine in a disclosed DNA sequence would be considered identical to the DNA sequence since both uracil and thymidine pair with adenine. Shortened and lengthened versions of the antisense compounds described herein as well as compounds having non-identical bases relative to the antisense compounds provided herein also are contemplated. The non-identical bases may be adjacent to each other or dispersed throughout the antisense compound. Percent identity of an antisense compound is calculated according to the number of bases that have identical base pairing relative to the sequence to which it is being compared.

In certain embodiments, the antisense compounds, or portions thereof, are at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to one or more of the antisense compounds or SEQ ID NOs, or a portion thereof, disclosed herein.

In certain embodiments, a portion of the antisense compound is compared to an equal length portion of the target nucleic acid. In certain embodiments, an 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 nucleobase portion is compared to an equal length portion of the target nucleic acid.

In certain embodiments, a portion of the antisense oligonucleotide is compared to an equal length portion of the target nucleic acid. In certain embodiments, an 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 nucleobase portion is compared to an equal length portion of the target nucleic acid.

Modifications

A nucleoside is a base-sugar combination. The nucleobase (also known as base) portion of the nucleoside is normally a heterocyclic base moiety. Nucleotides are nucleosides that further include a phosphate group covalently linked to the sugar portion of the nucleoside. For those nucleosides that include a pentofuranosyl sugar, the phosphate group can be linked to the 2′, 3′ or 5′ hydroxyl moiety of the sugar. Oligonucleotides are formed through the covalent linkage of adjacent nucleosides to one another, to form a linear polymeric oligonucleotide. Within the oligonucleotide structure, the phosphate groups are commonly referred to as forming the internucleoside linkages of the oligonucleotide.

Modifications to antisense compounds encompass substitutions or changes to internucleoside linkages, sugar moieties, or nucleobases. Modified antisense compounds are often preferred over native forms because of desirable properties such as, for example, enhanced cellular uptake, enhanced affinity for nucleic acid target, increased stability in the presence of nucleases, or increased inhibitory activity.

Chemically modified nucleosides may also be employed to increase the binding affinity of a shortened or truncated antisense oligonucleotide for its target nucleic acid. Consequently, comparable results can often be obtained with shorter antisense compounds that have such chemically modified nucleosides.

Modified Internucleoside Linkages

The naturally occurring internucleoside linkage of RNA and DNA is a 3′ to 5′ phosphodiester linkage. Antisense compounds having one or more modified, i.e. non-naturally occurring, internucleoside linkages are often selected over antisense compounds having naturally occurring internucleoside linkages because of desirable properties such as, for example, enhanced cellular uptake, enhanced affinity for target nucleic acids, and increased stability in the presence of nucleases.

Oligonucleotides having modified internucleoside linkages include internucleoside linkages that retain a phosphorus atom as well as internucleoside linkages that do not have a phosphorus atom. Representative phosphorus containing internucleoside linkages include, but are not limited to, phosphodiesters, phosphotriesters, methylphosphonates, phosphoramidate, and phosphorothioates. Methods of preparation of phosphorous-containing and non-phosphorous-containing linkages are well known.

In certain embodiments, antisense compounds targeted to a Factor 11 nucleic acid comprise one or more modified internucleoside linkages. In certain embodiments, the modified internucleoside linkages are phosphorothioate linkages. In certain embodiments, each internucleoside linkage of an antisense compound is a phosphorothioate internucleoside linkage.

Modified Sugar Moieties

Antisense compounds of the invention can optionally contain one or more nucleosides wherein the sugar group has been modified. Such sugar modified nucleosides may impart enhanced nuclease stability, increased binding affinity or some other beneficial biological property to the antisense compounds. In certain embodiments, nucleosides comprise a chemically modified ribofuranose ring moieties. Examples of chemically modified ribofuranose rings include without limitation, addition of substitutent groups (including 5′ and 2′ substituent groups, bridging of non-geminal ring atoms to form bicyclic nucleic acids (BNA), replacement of the ribosyl ring oxygen atom with S, N(R), or C(R1)(R)2 (R═H, C1-C12 alkyl or a protecting group) and combinations thereof. Examples of chemically modified sugars include 2′-F-5′-methyl substituted nucleoside (see PCT International Application WO 2008/101157 Published on Aug. 21, 2008 for other disclosed 5′,2′-bis substituted nucleosides) or replacement of the ribosyl ring oxygen atom with S with further substitution at the 2′-position (see published U.S. Patent Application US2005-0130923, published on Jun. 16, 2005) or alternatively 5′-substitution of a BNA (see PCT International Application WO 2007/134181 Published on Nov. 22, 2007 wherein LNA is substituted with for example a 5′-methyl or a 5′-vinyl group).

Examples of nucleosides having modified sugar moieties include without limitation nucleosides comprising 5′-vinyl, 5′-methyl (R or S), 4′-S, 2′-F, 2′-OCH3 and 2′-O(CH2)2PCH3 substituent groups. The substituent at the 2′ position can also be selected from allyl, amino, azido, thio, O-allyl, O—C1-C10 alkyl, OCF3, O(CH2)2SCH3, O(CH2)2-O—N(Rm)(Rn), and O-CH2—C(═O)—N(Rm)(Rn), where each Rm and Rn is, independently, H or substituted or unsubstituted C1-C10 alkyl.

Examples of bicyclic nucleic acids (BNAs) include without limitation nucleosides comprising a bridge between the 4′ and the 2′ ribosyl ring atoms. In certain embodiments, antisense compounds provided herein include one or more BNA nucleosides wherein the bridge comprises one of the formulas: 4′-(CH2)-O-2′ (LNA); 4′-(CH2)-S-2; 4′-(CH2)-O-2′ (LNA); 4′-(CH2)2-O-2′ (ENA); 4′-C(CH3)2-O-2′ (see PCT/US2008/068922); 4′-CH(CH3)

-O-2′ and 4′-C

H(CH₂OCH3)

-O-2′ (see U.S. Pat. No. 7,399,845, issued on Jul. 15, 2008); 4′-CH2—N(OCH3)-2′ (see PCT/US2008/064591); 4′-CH2-O—N(CH3)-2′ (see published U.S. Patent Application US2004-0171570, published Sep. 2, 2004); 4′-CH2—N(R)—O-2′ (see U.S. Pat. No. 7,427,672, issued on Sep. 23, 2008); 4′-CH2—C(CH3)-2′ and 4′-CH2—C

(═CH2)-2′ (see PCT/US2008/066154); and wherein R is, independently, H, C1-C12 alkyl, or a protecting group. Each of the foregoing BNAs include various stereochemical sugar configurations including for example α-L-ribofuranose and β-D-ribofuranose (see PCT international application PCT/DK98/00393, published on Mar. 25, 1999 as WO 99/14226).

In certain embodiments, nucleosides are modified by replacement of the ribosyl ring with a sugar surrogate. Such modification includes without limitation, replacement of the ribosyl ring with a surrogate ring system (sometimes referred to as DNA analogs) such as a morpholino ring, a cyclohexenyl ring, a cyclohexyl ring or a tetrahydropyranyl ring such as one having one of the formula:

Many other bicyclo and tricyclo sugar surrogate ring systems are also know in the art that can be used to modify nucleosides for incorporation into antisense compounds (see for example review article: Leumann, J. C, Bioorganic & Medicinal Chemistry, 2002, 10, 841-854). Such ring systems can undergo various additional substitutions to enhance activity.

Methods for the preparations of modified sugars are well known to those skilled in the art.

In nucleotides having modified sugar moieties, the nucleobase moieties (natural, modified or a combination thereof) are maintained for hybridization with an appropriate nucleic acid target.

In certain embodiments, antisense compounds targeted to a Factor 11 nucleic acid comprise one or more nucleotides having modified sugar moieties. In certain embodiments, the modified sugar moiety is 2′-MOE. In certain embodiments, the 2′-MOE modified nucleotides are arranged in a gapmer motif.

Modified Nucleobases

Nucleobase (or base) modifications or substitutions are structurally distinguishable from, yet functionally interchangeable with, naturally occurring or synthetic unmodified nucleobases. Both natural and modified nucleobases are capable of participating in hydrogen bonding. Such nucleobase modifications may impart nuclease stability, binding affinity or some other beneficial biological property to antisense compounds. Modified nucleobases include synthetic and natural nucleobases such as, for example, 5-methylcytosine (5-me-C). Certain nucleobase substitutions, including 5-methylcytosine substitutions, are particularly useful for increasing the binding affinity of an antisense compound for a target nucleic acid. For example, 5-methylcytosine substitutions have been shown to increase nucleic acid duplex stability by 0.6-1.2° C. (Sanghvi, Y. S., Crooke, S. T. and Lebleu, B., eds., Antisense Research and Applications, CRC Press, Boca Raton, 1993, pp. 276-278).

Additional modified nucleobases include 5-hydroxymethyl cytosine, xanthine, hypoxanthine, 2-aminoadenine, 6-methyl and other alkyl derivatives of adenine and guanine, 2-propyl and other alkyl derivatives of adenine and guanine, 2-thiouracil, 2-thiothymine and 2-thiocytosine, 5-halouracil and cytosine, 5-propynyl (—C≡C—CH₃) uracil and cytosine and other alkynyl derivatives of pyrimidine bases, 6-azo uracil, cytosine and thymine, 5-uracil (pseudouracil), 4-thiouracil, 8-halo, 8-amino, 8-thiol, 8-thioalkyl, 8-hydroxyl and other 8-substituted adenines and guanines, 5-halo particularly 5-bromo, 5-trifluoromethyl and other 5-substituted uracils and cytosines, 7-methylguanine and 7-methyladenine, 2-F-adenine, 2-amino-adenine, 8-azaguanine and 8-azaadenine, 7-deazaguanine and 7-deazaadenine and 3-deazaguanine and 3-deazaadenine.

Heterocyclic base moieties may also include those in which the purine or pyrimidine base is replaced with other heterocycles, for example 7-deaza-adenine, 7-deazaguanosine, 2-aminopyridine and 2-pyridone. Nucleobases that are particularly useful for increasing the binding affinity of antisense compounds include 5-substituted pyrimidines, 6-azapyrimidines and N-2, N-6 and O-6 substituted purines, including 2 aminopropyladenine, 5-propynyluracil and 5-propynylcytosine.

In certain embodiments, antisense compounds targeted to a Factor 11 nucleic acid comprise one or more modified nucleobases. In certain embodiments, gap-widened antisense oligonucleotides targeted to a Factor 11 nucleic acid comprise one or more modified nucleobases. In certain embodiments, the modified nucleobase is 5-methylcytosine. In certain embodiments, each cytosine is a 5-methylcytosine.

Compositions and Methods for Formulating Pharmaceutical Compositions

Antisense oligonucleotides may be admixed with pharmaceutically acceptable active or inert substances for the preparation of pharmaceutical compositions or formulations. Compositions and methods for the formulation of pharmaceutical compositions are dependent upon a number of criteria, including, but not limited to, route of administration, extent of disease, or dose to be administered.

An antisense compound targeted to a Factor 11 nucleic acid can be utilized in pharmaceutical compositions by combining the antisense compound with a suitable pharmaceutically acceptable diluent or carrier. A pharmaceutically acceptable diluent includes phosphate-buffered saline (PBS). PBS is a diluent suitable for use in compositions to be delivered parenterally. Accordingly, in one embodiment, employed in the methods described herein is a pharmaceutical composition comprising an antisense compound targeted to a Factor 11 nucleic acid and a pharmaceutically acceptable diluent. In certain embodiments, the pharmaceutically acceptable diluent is PBS. In certain embodiments, the antisense compound is an antisense oligonucleotide.

Pharmaceutical compositions comprising antisense compounds encompass any pharmaceutically acceptable salts, esters, or salts of such esters, or any other oligonucleotide which, upon administration to an animal, including a human, is capable of providing (directly or indirectly) the biologically active metabolite or residue thereof. Accordingly, for example, the disclosure is also drawn to pharmaceutically acceptable salts of antisense compounds, prodrugs, pharmaceutically acceptable salts of such prodrugs, and other bioequivalents. Suitable pharmaceutically acceptable salts include, but are not limited to, sodium and potassium salts.

A prodrug can include the incorporation of additional nucleosides at one or both ends of an antisense compound which are cleaved by endogenous nucleases within the body, to form the active antisense compound.

Conjugated Antisense Compounds

Antisense compounds may be covalently linked to one or more moieties or conjugates which enhance the activity, cellular distribution or cellular uptake of the resulting antisense oligonucleotides. Typical conjugate groups include cholesterol moieties and lipid moieties. Additional conjugate groups include carbohydrates, phospholipids, biotin, phenazine, folate, phenanthridine, anthraquinone, acridine, fluoresceins, rhodamines, coumarins, and dyes.

Antisense compounds can also be modified to have one or more stabilizing groups that are generally attached to one or both termini of antisense compounds to enhance properties such as, for example, nuclease stability. Included in stabilizing groups are cap structures. These terminal modifications protect the antisense compound having terminal nucleic acid from exonuclease degradation, and can help in delivery and/or localization within a cell. The cap can be present at the 5′-terminus (5′-cap), or at the 3′-terminus (3′-cap), or can be present on both termini. Cap structures are well known in the art and include, for example, inverted deoxy abasic caps. Further 3′ and 5′-stabilizing groups that can be used to cap one or both ends of an antisense compound to impart nuclease stability include those disclosed in WO 03/004602 published on Jan. 16, 2003.

Cell Culture and Antisense Compounds Treatment

The effects of antisense compounds on the level, activity or expression of Factor 11 nucleic acids can be tested in vitro in a variety of cell types. Cell types used for such analyses are available from commerical vendors (e.g. American Type Culture Collection, Manassus, Va.; Zen-Bio, Inc., Research Triangle Park, N.C.; Clonetics Corporation, Walkersville, Md.) and are cultured according to the vendor's instructions using commercially available reagents (e.g. Invitrogen Life Technologies, Carlsbad, Calif.). Illustrative cell types include, but are not limited to, HepG2 cells, Hep3B cells, and primary hepatocytes.

In Vitro Testing of Antisense Oligonucleotides

Described herein are methods for treatment of cells with antisense oligonucleotides, which can be modified appropriately for treatment with other antisense compounds.

In general, cells are treated with antisense oligonucleotides when the cells reach approximately 60-80% confluency in culture.

One reagent commonly used to introduce antisense oligonucleotides into cultured cells includes the cationic lipid transfection reagent LIPOFECTIN (Invitrogen, Carlsbad, Calif.). Antisense oligonucleotides are mixed with LIPOFECTIN in OPTI-MEM 1 (Invitrogen, Carlsbad, Calif.) to achieve the desired final concentration of antisense oligonucleotide and a LIPOFECTIN concentration that typically ranges 2 to 12 ug/mL per 100 nM antisense oligonucleotide.

Another reagent used to introduce antisense oligonucleotides into cultured cells includes LIPOFECTAMINE (Invitrogen, Carlsbad, Calif.). Antisense oligonucleotide is mixed with LIPOFECTAMINE in OPTI-MEM 1 reduced serum medium (Invitrogen, Carlsbad, Calif.) to achieve the desired concentration of antisense oligonucleotide and a LIPOFECTAMINE concentration that typically ranges 2 to 12 ug/mL per 100 nM antisense oligonucleotide.

Another technique used to introduce antisense oligonucleotides into cultured cells includes electroporation.

Cells are treated with antisense oligonucleotides by routine methods. Cells are typically harvested 16-24 hours after antisense oligonucleotide treatment, at which time RNA or protein levels of target nucleic acids are measured by methods known in the art and described herein. In general, when treatments are performed in multiple replicates, the data are presented as the average of the replicate treatments.

The concentration of antisense oligonucleotide used varies from cell line to cell line. Methods to determine the optimal antisense oligonucleotide concentration for a particular cell line are well known in the art. Antisense oligonucleotides are typically used at concentrations ranging from 1 nM to 300 nM when transfected with LIPOFECTAMINE. Antisense oligonucleotides are used at higher concentrations ranging from 625 to 20,000 nM when transfected using electroporation.

RNA Isolation

RNA analysis can be performed on total cellular RNA or poly(A)+ mRNA. Methods of RNA isolation are well known in the art. RNA is prepared using methods well known in the art, for example, using the TRIZOL Reagent (Invitrogen, Carlsbad, Calif.) according to the manufacturer's recommended protocols.

Analysis of Inhibition of Target Levels or Expression

Inhibition of levels or expression of a Factor 11 nucleic acid can be assayed in a variety of ways known in the art. For example, target nucleic acid levels can be quantitated by, e.g., Northern blot analysis, competitive polymerase chain reaction (PCR), or quantitaive real-time PCR. RNA analysis can be performed on total cellular RNA or poly(A)+ mRNA. Methods of RNA isolation are well known in the art. Northern blot analysis is also routine in the art. Quantitative real-time PCR can be conveniently accomplished using the commercially available ABI PRISM 7600, 7700, or 7900 Sequence Detection System, available from PE-Applied Biosystems, Foster City, Calif. and used according to manufacturer's instructions.

Quantitative Real-Time PCR Analysis of Target RNA Levels

Quantitation of target RNA levels may be accomplished by quantitative real-time PCR using the ABI PRISM 7600, 7700, or 7900 Sequence Detection System (PE-Applied Biosystems, Foster City, Calif.) according to manufacturer's instructions. Methods of quantitative real-time PCR are well known in the art.

Prior to real-time PCR, the isolated RNA is subjected to a reverse transcriptase (RT) reaction, which produces complementary DNA (cDNA) that is then used as the substrate for the real-time PCR amplification. The RT and real-time PCR reactions are performed sequentially in the same sample well. RT and real-time PCR reagents are obtained from Invitrogen (Carlsbad, Calif.). RT real-time-PCR reactions are carried out by methods well known to those skilled in the art.

Gene (or RNA) target quantities obtained by real time PCR are normalized using either the expression level of a gene whose expression is constant, such as cyclophilin A, or by quantifying total RNA using RIBOGREEN (Invitrogen, Inc. Carlsbad, Calif.). Cyclophilin A expression is quantified by real time PCR, by being run simultaneously with the target, multiplexing, or separately. Total RNA is quantified using RIBOGREEN RNA quantification reagent (Invetrogen, Inc. Eugene, Oreg.). Methods of RNA quantification by RIBOGREEN are taught in Jones, L. J., et al, (Analytical Biochemistry, 1998, 265, 368-374). A CYTOFLUOR 4000 instrument (PE Applied Biosystems) is used to measure RIBOGREEN fluorescence.

Probes and primers are designed to hybridize to a Factor 11 nucleic acid. Methods for designing real-time PCR probes and primers are well known in the art, and may include the use of software such as PRIMER EXPRESS Software (Applied Biosystems, Foster City, Calif.).

Analysis of Protein Levels

Antisense inhibition of Factor 11 nucleic acids can be assessed by measuring Factor 11 protein levels. Protein levels of Factor 11 can be evaluated or quantitated in a variety of ways well known in the art, such as immunoprecipitation, Western blot analysis (immunoblotting), enzyme-linked immunosorbent assay (ELISA), quantitative protein assays, protein activity assays (for example, caspase activity assays), immunohistochemistry, immunocytochemistry or fluorescence-activated cell sorting (FACS). Antibodies directed to a target can be identified and obtained from a variety of sources, such as the MSRS catalog of antibodies (Aerie Corporation, Birmingham, Mich.), or can be prepared via conventional monoclonal or polyclonal antibody generation methods well known in the art. Antibodies useful for the detection of mouse, rat, monkey, and human Factor 11 are commercially available.

In Vivo Testing of Antisense Compounds

Antisense compounds, for example, antisense oligonucleotides, are tested in animals to assess their ability to inhibit expression of Factor 11 and produce phenotypic changes, such as, prolonged aPTT, prolonged aPTT time in conjunction with a normal PT, decreased quantity of Platelet Factor 4 (PF-4), and reduced formation of thrombus or increased time for thrombus formation. Testing may be performed in normal animals, or in experimental disease models. For administration to animals, antisense oligonucleotides are formulated in a pharmaceutically acceptable diluent, such as phosphate-buffered saline. Administration includes parenteral routes of administration, such as intraperitoneal, intravenous, and subcutaneous. Calculation of antisense oligonucleotide dosage and dosing frequency is within the abilities of those skilled in the art, and depends upon factors such as route of administration and animal body weight. Following a period of treatment with antisense oligonucleotides, RNA is isolated from liver tissue and changes in Factor 11 nucleic acid expression are measured. Changes in Factor 11 protein levels are also measured using a thrombin generation assay. In addition, effects on clot times, e.g. PT and aPTT, are determined using plasma from treated animals.

Tolerability

In certain embodiments, the compounds provided herein display minimal side effects. Side effects include responses to the administration of the antisense compound that are typically unrelated to the targeting of factor 11, such as an inflammatory response in the animal. In certain embodiments compounds are well tolerated by the animal. Increased tolerability can depend on a number of factors, including, but not limited to, the nucleotide sequence of the antisense compound, chemical modifications to the nucleotides, the particular motif of unmodified and modified nucleosides in the antisense compound, or combinations thereof. Tolerability may be determined by a number of factors. Such factors include body weight, organ weight, liver function, kidney function, platelet count, white blood cell count.

In certain embodiments, the compounds provided herein demonstrate minimal effect on organ weight. In certain embodiments, the compounds demonstrate less than a 7-fold, 6-fold, 5-fold, 4-fold, 3-fold, 2-fold or no significant increase in spleen and/or liver weight.

In certain embodiments, the compounds provided herein demonstrate minimal effect on liver function. Factors for the evaluation of liver function include ALT levels, AST levels, plasma bilirubin levels and plasma albumin levels. In certain embodiments the compounds provided herein demonstrate less than a 7-fold, less than a 6-fold, less than a 5-fold, less than a 4-fold, less than a 3-fold or less than a 2-fold or no significant increase in ALT or AST. In certain embodiments the compounds provided herein demonstrate less than a 3-fold, less than a 2-fold or no significant increase in plasma bilirubin levels.

In certain embodiments, the compounds provided herein demonstrate minimal effect on kidney function. In certain embodiments, the compounds provided herein demonstrate less than a 3-fold, less than a 2-fold, or no significant increase in plasma concentrations of blood urea nitrogen (BUN). In certain embodiments, the compounds provided herein demonstrate less than a 6-fold, 5-fold, 4-fold, 3-fold, 2-fold, or no significant increase in the ratio of urine protein to creatinine.

In certain embodiments, the compounds provided herein demonstrate minimal effect on hematological factors. In certain embodiments, the compounds provided herein demonstrate less than a 60%, 50%, 40%, 30%, 20%, 10% or 5% decrease in platelet count. In certain embodiments, the compounds provided herein demonstrate less than a 4-fold, less than a 3-fold, less than a 2-fold or no significant increase in monocyte count.

In certain embodiments compounds further display favorable pharmacokinetics. In certain embodiments, antisense compounds exhibit relatively high half-lives in relevant biological fluids or tissues.

In certain embodiments, compounds or compositions further display favorable viscosity. In certain embodiments, the viscosity of the compound or composition is no more than 40 cP at a concentration of 165-185 mg/mL.

In other embodiments, the compounds display combinations of the characteristics above and reduce factor 11 mRNA expression in an animal model with high efficiency.

Certain Indications

In certain embodiments, the invention provides methods of treating an individual comprising administering one or more pharmaceutical compositions of the present invention. In certain embodiments, the individual has a thromboembolic complication. In certain embodiments, the individual is at risk for a blood clotting disorder, including, but not limited to, infarct, thrombosis, embolism, thromboembolism such as deep vein thrombosis, pulmonary embolism, myocardial infarction, and stroke. This includes individuals with an acquired problem, disease, or disorder that leads to a risk of thrombosis, for example, surgery, cancer, immobility, sepsis, atherosclerosis atrial fibrillation, as well as genetic predisposition, for example, antiphospholipid syndrome and the autosomal dominant condition, Factor V Leiden. In certain embodiments, the individual has been identified as in need of anticoagulation therapy. Examples of such individuals include, but are not limited to, those undergoing major orthopedic surgery (e.g., hip/knee replacement or hip fracture surgery) and patients in need of chronic treatment, such as those suffering from arterial fibrillation to prevent stroke. In certain embodiments the invention provides methods for prophylactically reducing Factor 11 expression in an individual. Certain embodiments include treating an individual in need thereof by administering to an individual a therapeutically effective amount of an antisense compound targeted to a Factor 11 nucleic acid.

In one embodiment, administration of a therapeutically effective amount of an antisense compound targeted to a Factor 11 nucleic acid is accompanied by monitoring of Factor 11 levels in the serum of an individual, to determine an individual's response to administration of the antisense compound. An individual's response to administration of the antisense compound is used by a physician to determine the amount and duration of therapeutic intervention.

In certain embodiments, administration of an antisense compound targeted to a Factor 11 nucleic acid results in reduction of Factor 11 expression by at least 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95 or 99%, or a range defined by any two of these values. In certain embodiments, administration of an antisense compound targeted to a Factor 11 nucleic acid results in a change in a measure of blood clotting as measured by a standard test, for example, but not limited to, activated partial thromboplastin time (aPTT) test, prothrombin time (PT) test, thrombin time (TCT), bleeding time, or D-dimer. In certain embodiments, administration of a Factor 11 antisense compound increases the measure by at least 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95 or 99%, or a range defined by any two of these values. In some embodiments, administration of a Factor 11 antisense compound decreases the measure by at least 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95 or 99%, or a range defined by any two of these values.

In certain embodiments, pharmaceutical compositions comprising an antisense compound targeted to Factor 11 are used for the preparation of a medicament for treating a patient suffering or susceptible to a thromboembolic complication.

Certain Combination Therapies

In certain embodiments, one or more pharmaceutical compositions of the present invention are co-administered with one or more other pharmaceutical agents. In certain embodiments, such one or more other pharmaceutical agents are designed to treat the same disease, disorder, or condition as the one or more pharmaceutical compositions of the present invention. In certain embodiments, such one or more other pharmaceutical agents are designed to treat a different disease, disorder, or condition as the one or more pharmaceutical compositions of the present invention. In certain embodiments, such one or more other pharmaceutical agents are designed to treat an undesired side effect of one or more pharmaceutical compositions of the present invention. In certain embodiments, one or more pharmaceutical compositions of the present invention are co-administered with another pharmaceutical agent to treat an undesired effect of that other pharmaceutical agent. In certain embodiments, one or more pharmaceutical compositions of the present invention are co-administered with another pharmaceutical agent to produce a combinational effect. In certain embodiments, one or more pharmaceutical compositions of the present invention are co-administered with another pharmaceutical agent to produce a synergistic effect. In certain embodiments, one or more pharmaceutical compositions of the present invention and one or more other pharmaceutical agents are administered at the same time. In certain embodiments, one or more pharmaceutical compositions of the present invention and one or more other pharmaceutical agents are administered at different times. In certain embodiments, one or more pharmaceutical compositions of the present invention and one or more other pharmaceutical agents are prepared together in a single formulation. In certain embodiments, one or more pharmaceutical compositions of the present invention and one or more other pharmaceutical agents are prepared separately.

In certain embodiments, pharmaceutical agents that may be co-administered with a pharmaceutical composition of the present invention include anticoagulant or antiplatelet agents. In certain embodiments, pharmaceutical agents that may be co-administered with a pharmaceutical composition of the present invention include NSAID/Cyclooxygenase inhibitors, such as, aspirin. In certain embodiments, pharmaceutical agents that may be co-administered with a pharmaceutical composition of the present invention include adenosine diphosphate (ADP) receptor inhibitors, such as, clopidogrel (PLAVIX) and ticlopidine (TICLID). In certain embodiments, pharmaceutical agents that may be co-administered with a pharmaceutical composition of the present invention include phosphodiesterase inhibitors, such as, cilostazol (PLETAL). In certain embodiments, pharmaceutical agents that may be co-administered with a pharmaceutical composition of the present invention include, glycoprotein IIB/IIIA inhibitors, such as, abciximab (REOPRO), eptifibatide (INTEGRILIN), tirofiban (AGGRASTAT), and defibrotide. In certain embodiments, pharmaceutical agents that may be co-administered with a pharmaceutical composition of the present invention include, adenosine reuptake inhibitors, such as, to dipyridamole (PERSANTINE). In certain embodiments, pharmaceutical agents that may be co-administered with a pharmaceutical composition of the present invention include, but are not limited to warfarin (and related coumarins), heparin, direct thrombin inhibitors (such as lepirudin, bivalirudin), apixaban, LOVENOX, and small molecular compounds that interfere directly with the enzymatic action of particular coagulation factors (e.g. rivaroxaban, which interferes with Factor Xa). In certain embodiments, pharmaceutical agents that may be co-administered with a Factor 11 specific inhibitor of the present invention include, but are not limited to, an additional Factor 11 inhibitor. In certain embodiments, the anticoagulant or antiplatelet agent is administered prior to administration of a pharmaceutical composition of the present invention. In certain embodiments, the anticoagulant or antiplatelet agent is administered following administration of a pharmaceutical composition of the present invention. In certain embodiments the anticoagulant or antiplatelet agent is administered at the same time as a pharmaceutical composition of the present invention. In certain embodiments the dose of a co-administered anticoagulant or antiplatelet agent is the same as the dose that would be administered if the anticoagulant or antiplatelet agent was administered alone. In certain embodiments the dose of a co-administered anticoagulant or antiplatelet agent is lower than the dose that would be administered if the anticoagulant or antiplatelet agent was administered alone. In certain embodiments the dose of a co-administered anticoagulant or antiplatelet agent is greater than the dose that would be administered if the anticoagulant or antiplatelet agent was administered alone.

In certain embodiments, the co-administration of a second compound enhances the anticoagulant effect of a first compound, such that co-administration of the compounds results in an anticoagulant effect that is greater than the effect of administering the first compound alone. In other embodiments, the co-administration results in anticoagulant effects that are additive of the effects of the compounds when administered alone. In certain embodiments, the co-administration results in anticoagulant effects that are supra-additive of the effects of the compounds when administered alone. In certain embodiments, the co-administration of a second compound increases antithrombotic activity without increased bleeding risk. In certain embodiments, the first compound is an antisense compound. In certain embodiments, the second compound is an antisense compound.

In certain embodiments, an antidote is administered anytime after the administration of a Factor 11 specific inhibitor. In certain embodiments, an antidote is administered anytime after the administration of an antisense oligonucleotide targeting Factor 11. In certain embodiments, the antidote is administered minutes, hours, days, weeks, or months after the administration of an antisense compound targeting Factor 11. In certain embodiments, the antidote is a complementary (e.g. the sense strand) to the antisense compound targeting Factor 11. In certain embodiments, the antidote is a Factor 7, Factor 7a, Factor 11, or Factor 11a protein. In certain embodiments, the Factor 7, Factor 7a, Factor 11, or Factor 11a protein is a human Factor 7, human Factor 7a, human Factor 11, or human Factor 11a protein. In certain embodiments, the Factor 7 protein is NOVOSEVEN.

Certain Co-Administered Antiplatelet Therapies

In certain embodiments, Factor 11 inhibitors are combined with antiplatelet therapies. In certain embodiments, administration of a Factor 11 inhibitor in combination with an antiplatelet therapy results in little to no appreciable or detectable increase in risk of bleeding as compared to antiplatelet therapy alone. In certain embodiments, the risk profile or risk indications are unchanged over antiplatelet therapy alone.

The combination of antiplatelet and anticoagulant therapy is used in clinical practice most frequently in patients diagnosed with, for example, thromboembolism, atrial fibrillation, a heart valve disorder, valvular heart disease, stroke, CAD, and in patients having a mechanical valve. The benefit of dual therapy relates to the probable additive effect of suppressing both platelet and coagulation factor activities. The risk of dual therapy is the potential for increased bleeding (Dowd, M. Plenary Sessions/Thrombosis Research 123 (2008)).

Prior combinations of antiplatelet and anticoagulant therapy have been shown to increase the risk of bleeding compared with anticoagulant or antiplatelet therapy alone. Such combinations include, FXa inhibitors (e.g., apixiban and rivaroxaban) with ADP receptor/P2Y12 inhibitors (Thienopyridines such as clopidogrel—also known as PLAVIX) and NSAIDs (e.g., aspirin and naproxen) (Kubitza, D. et al., Br. J. Clin. Pharmacol. 63:4 (2006); Wong, P. C. et al. Journal of Thrombosis and Haemostasis 6 (2008); FDA Advisory Committee Briefing Document for New Drug Application 22-406 (2009)). For example, Wong reports that addition of certain doses of apixaban to aspirin and to aspirin plus clopidogrel produced a significant increase in bleeding time compared with aspirin alone and asprin plus clopidogrel. Kubitza reports that the combination administration of rivaroxaban and naproxen significantly increased bleeding time over naproxen alone.

EXAMPLES Non-Limiting Disclosure and Incorporation by Reference

While certain compounds, compositions and methods described herein have been described with specificity in accordance with certain embodiments, the following examples serve only to illustrate the compounds described herein and are not intended to limit the same. Each of the references recited in the present application is incorporated herein by reference in its entirety.

Example 1 Antisense Inhibition of Human Factor 11 in HepG2 Cells

Antisense oligonucleotides targeted to a Factor 11 nucleic acid were tested for their effects on Factor 11 mRNA in vitro. Cultured HepG2 cells at a density of 10,000 cells per well were transfected using lipofectin reagent with 75 nM antisense oligonucleotide. After a treatment period of approximately 24 hours, RNA was isolated from the cells and Factor 11 mRNA levels were measured by quantitative real time PCR. Factor 11 mRNA levels were adjusted according to total RNA content, as measured by RIBOGREEN. Results are presented as percent inhibition of Factor 11, relative to untreated control cells.

The chimeric antisense oligonucleotides in Tables 1 and 2 were designed as 5-10-5 MOE gapmers. The gapmers are 20 nucleotides in length, wherein the central gap segment is comprised of 10 2′-deoxynucleotides and is flanked on both sides (in the 5′ and 3′ directions) by wings comprising 5 nucleotides each. Each nucleotide in the 5′ wing segment and each nucleotide in the 3′ wing segment has a 2′-MOE modification. The internucleoside linkages throughout each gapmer are phosphorothioate (P═S) linkages. All cytidine residues throughout each gapmer are 5-methylcytidines. “Target start site” indicates the 5′-most nucleotide to which the gapmer is targeted. “Target stop site” indicates the 3′-most nucleotide to which the gapmer is targeted. Each gapmer listed in Table 1 is targeted to SEQ ID NO: 1 (GENBANK Accession No. NM_(—)000128.3) and each gapmer listed in Table 2 is targeted to SEQ ID NO: 2 (GENBANK Accession No. NT_(—)022792.17, truncated from 19598000 to 19624000).

TABLE 1 Inhibition of human Factor 11 mRNA levels by chimeric antisense oligonucleotides having 5-10-5 MOE wings and deoxy gap targeted to SEQ ID NO: 1 Target Target Oligo Start Stop SEQ ID ID Site Site Sequence % inhibition NO 412187 38 57 TTCAAACAAGTGACATACAC 21 15 412188 96 115 TGAGAGAATTGCTTGCTTTC 21 16 412189 106 125 AAATATACCTTGAGAGAATT 8 17 412190 116 135 AGTATGTCAGAAATATACCT 24 18 412191 126 145 TTAAAATCTTAGTATGTCAG 14 19 412192 146 165 CAGCATATTTGTGAAAGTCG 44 20 412193 222 241 TGTGTAGGAAATGGTCACTT 38 21 412194 286 305 TGCAATTCTTAATAAGGGTG 80 22 412195 321 340 AAATCATCCTGAAAAGACCT 22 23 412196 331 350 TGATATAAGAAAATCATCCT 25 24 412197 376 395 ACACATTCACCAGAAACTGA 45 25 412198 550 569 TTCAGGACACAAGTAAACCA 21 26 412199 583 602 TTCACTCTTGGCAGTGTTTC 66 27 412200 612 631 AAGAATACCCAGAAATCGCT 59 28 412201 622 641 CATTGCTTGAAAGAATACCC 66 29 412202 632 651 TTGGTGTGAGCATTGCTTGA 65 30 412203 656 675 AATGTCTTTGTTGCAAGCGC 91 31 412204 676 695 TTCATGTCTAGGTCCACATA 74 32 412205 686 705 GTTTATGCCCTTCATGTCTA 69 33 412206 738 757 CCGTGCATCTTTCTTGGCAT 87 34 412207 764 783 CGTGAAAAAGTGGCAGTGGA 64 35 412208 811 830 AGACAAATGTTACGATGCTC 73 36 412209 821 840 GTGCTTCAGTAGACAAATGT 91 37 412210 896 915 TGCACAGGATTTCAGTGAAA 73 38 412211 906 925 GATTAGAAAGTGCACAGGAT 64 39 412212 1018 1037 CCGGGATGATGAGTGCAGAT 88 40 412213 1028 1047 AAACAAGCAACCGGGATGAT 71 41 412214 1048 1067 TCCTGGGAAAAGAAGGTAAA 58 42 412215 1062 1081 ATTCTTTGGGCCATTCCTGG 81 43 412216 1077 1096 AAAGATTTCTTTGAGATTCT 43 44 412217 1105 1124 AATCCACTCTCAGATGTTTT 47 45 412218 1146 1165 AACCAGAAAGAGCTTTGCTC 27 46 412219 1188 1207 GGCAGAACACTGGGATGCTG 56 47 412220 1204 1223 TGGTAAAATGAAGAATGGCA 58 48 412221 1214 1233 ATCAGTGTCATGGTAAAATG 48 49 412222 1241 1263 AACAATATCCAGTTCTTCTC 5 50 412223 1275 1294 ACAGTTTCTGGCAGGCCTCG 84 51 412224 1285 1304 GCATTGGTGCACAGTTTCTG 87 52 412225 1295 1314 GCAGCGGACGGCATTGGTGC 86 53 412226 1371 1390 TTGAAGAAAGCTTTAAGTAA 17 54 412227 1391 1410 AGTATTTTAGTTGGAGATCC 75 55 412228 1425 1444 ATGTGTATCCAGAGATGCCT 71 56 412229 1456 1475 GTACACTCATTATCCATTTT 64 57 412230 1466 1485 GATTTTGGTGGTACACTCAT 52 58 412231 1476 1495 TCCTGGGCTTGATTTTGGTG 74 59 412232 1513 1532 GGCCACTCACCACGAACAGA 80 60 412233 1555 1574 TGTCTCTGAGTGGGTGAGGT 64 61 412234 1583 1602 GTTTCCAATGATGGAGCCTC 60 62 412235 1593 1612 ATATCCACTGGTTTCCAATG 57 63 412236 1618 1637 CCATAGAAACAGTGAGCGGC 72 64 412237 1628 1647 TGACTCTACCCCATAGAAAC 48 65 412238 1642 1661 CGCAAAATCTTAGGTGACTC 71 66 412239 1673 1692 TTCAGATTGATTTAAAATGC 43 67 412240 1705 1724 TGAACCCCAAAGAAAGATGT 32 68 412241 1715 1734 TATTATTTCTTGAACCCCAA 41 69 412242 1765 1784 AACAAGGCAATATCATACCC 49 70 412243 1775 1794 TTCCAGTTTCAACAAGGCAA 70 71 412244 1822 1841 GAAGGCAGGCATATGGGTCG 53 72 412245 1936 1955 GTCACTAAGGGTATCTTGGC 75 73 412246 1992 2011 AGATCATCTTATGGGTTATT 68 74 412247 2002 2021 TAGCCGGCACAGATCATCTT 75 75 412248 2082 2101 CCAGATGCCAGACCTCATTG 53 76 412249 2195 2214 CATTCACACTGCTTGAGTTT 55 77 412250 2268 2287 TGGCACAGTGAACTCAACAC 63 78 412251 2326 2345 CTAGCATTTTCTTACAAACA 58 79 412252 2450 2469 TTATGGTAATTCTTGGACTC 39 80 412253 2460 2479 AAATATTGCCTTATGGTAAT 20 81 412254 2485 2504 TATCTGCCTATATAGTAATC 16 82 412255 2510 2529 GCCACTACTTGGTTATTTTC 38 83 412256 2564 2583 AACAAATCTATTTATGGTGG 39 84 412257 2622 2641 CTGCAAAATGGTGAAGACTG 57 85 412258 2632 2651 GTGTAGATTCCTGCAAAATG 44 86 412259 2882 2901 TTTTCAGGAAAGTGTATCTT 37 87 412260 2892 2911 CACAAATCATTTTTCAGGAA 27 88 412261 2925 2944 TCCCAAGATATTTTAAATAA 3 89 412262 3168 3187 AATGAGATAAATATTTGCAC 34 90 412263 3224 3243 TGAAAGCTATGTGGTGACAA 33 91 412264 3259 3278 CACACTTGATGAATTGTATA 27 92 413460 101 120 TACCTTGAGAGAATTGCTTG 40 93 413461 111 130 GTCAGAAATATACCTTGAGA 39 94 413462 121 140 ATCTTAGTATGTCAGAAATA 12 95 413463 381 400 GAGTCACACATTCACCAGAA 74 96 413464 627 646 GTGAGCATTGCTTGAAAGAA 42 97 413465 637 656 CTTATTTGGTGTGAGCATTG 80 98 413466 661 680 ACATAAATGTCTTTGTTGCA 79 99 413467 666 685 GGTCCACATAAATGTCTTTG 91 100 413468 671 690 GTCTAGGTCCACATAAATGT 84 101 413469 681 700 TGCCCTTCATGTCTAGGTCC 84 102 413470 692 711 GTTATAGTTTATGCCCTTCA 72 103 413471 816 835 TCAGTAGACAAATGTTACGA 67 104 413472 826 845 TGGGTGTGCTTCAGTAGACA 99 105 413473 911 930 AGCCAGATTAGAAAGTGCAC 80 106 413474 1023 1042 AGCAACCGGGATGATGAGTG 84 107 413475 1053 1072 GCCATTCCTGGGAAAAGAAG 80 108 413476 1067 1086 TTGAGATTCTTTGGGCCATT 88 109 413477 1151 1170 ACTGAAACCAGAAAGAGCTT 54 110 413478 1193 1212 AGAATGGCAGAACACTGGGA 53 111 413479 1209 1228 TGTCATGGTAAAATGAAGAA 40 112 413480 1219 1238 AAGAAATCAGTGTCATGGTA 71 113 413481 1280 1299 GGTGCACAGTTTCTGGCAGG 86 114 413482 1290 1309 GGACGGCATTGGTGCACAGT 85 115 413483 1300 1319 AACTGGCAGCGGACGGCATT 78 116 413484 1430 1449 CCTTAATGTGTATCCAGAGA 74 117 413485 1461 1480 TGGTGGTACACTCATTATCC 68 118 413486 1471 1490 GGCTTGATTTTGGTGGTACA 83 119 413487 1481 1500 AACGATCCTGGGCTTGATTT 57 120 413488 1560 1579 ACAGGTGTCTCTGAGTGGGT 49 121 413489 1588 1607 CACTGGTTTCCAATGATGGA 68 122 413490 1623 1642 CTACCCCATAGAAACAGTGA 57 123 413491 1633 1652 TTAGGTGACTCTACCCCATA 73 124 413492 1647 1666 AGAGACGCAAAATCTTAGGT 68 125 413493 1710 1729 TTTCTTGAACCCCAAAGAAA 65 126 413494 1780 1799 GTGGTTTCCAGTTTCAACAA 70 127 413495 1921 1940 TTGGCTTTCTGGAGAGTATT 58 128 413496 1997 2016 GGCACAGATCATCTTATGGG 72 129 413497 2627 2646 GATTCCTGCAAAATGGTGAA 39 130 413498 2637 2656 GCAGAGTGTAGATTCCTGCA 60 131 413499 2887 2906 ATCATTTTTCAGGAAAGTGT 52 132

TABLE 2 Inhibition of human Factor 11 mRNA levels by chimeric antisense oligonucleotides having 5-10-5 MOE wings and deoxy gap targeted to SEQ ID NO: 2 Target Target Oligo Start Stop SEQ ID ID Site Site Sequence % inhibition NO 413500 1658 1677 GTGAGACAAATCAAGACTTC 15 133 413501 2159 2178 TTAGTTTACTGACACTAAGA 23 134 413502 2593 2612 CTGCTTTATGAAAAACCAAC 22 135 413503 3325 3344 ATACCTAGTACAATGTAAAT 29 136 413504 3548 3567 GGCTTGTGTGTGGTCAATAT 54 137 413505 5054 5073 TGGGAAAGCTTTCAATATTC 57 138 413506 6474 6493 ATGGAATTGTGCTTATGAGT 57 139 413507 7590 7609 TTTCAAGCTCAGGATGGGAA 55 140 413508 7905 7924 GTTGGTAAAATGCAACCAAA 64 141 413509 8163 8182 TCAGGACACAAGTAAACCTG 66 142 413510 9197 9216 TGCAAGCTGGAAATAAAAGC 17 143 413511 9621 9640 TGCCAATTTAAAAGTGTAGC 43 144 413512 9800 9819 ATATTTCAAAATCCAGTATG 39 145 413513 9919 9938 TTCTGAATATACAAATTAAT 27 146 413514 9951 9970 TTTACTATGAAAATCTAAAT 5 147 413515 11049 11068 GGTATCCTGAGTGAGATCTA 36 148 413516 11269 11288 CCAGCTATCAGGAAAATTCC 50 149 413517 12165 12184 AAAGCTATTGGAGACTCAGA 51 150 413518 12584 12603 ATGGAATCTCTTCATTTCAT 49 151 413519 12728 12747 ATGGAGACATTCATTTCCAC 59 152 413520 13284 13303 GCTCTGAGAGTTCCAATTCA 52 153 413521 14504 14523 CTGGGAAGGTGAATTTTTAG 62 154 413522 14771 14790 TCAAGAGTCTTCATGCTACC 42 155 413523 15206 15225 TCAGTTTACCTGGGATGCTG 61 156 413524 15670 15689 GACATTATACTCACCATTAT 7 157 413525 15905 15924 GTATAAATGTGTCAAATTAA 43 158 413526 16482 16501 GTAAAGTTTTACCTTAACCT 47 159 413527 17298 17317 CCATAATGAAGAAGGAAGGG 52 160 413528 17757 17776 TTAAGTTACATTGTAGACCA 48 161 413529 18204 18223 TGTGTGGGTCCTGAAATTCT 52 162 413530 18981 19000 ATCTTGTAATTACACACCCC 27 163 413531 19174 19193 GTACACTCTGCAACAGAAGC 47 164 413532 19604 19623 AGGGAATAACATGAAGGCCC 32 165 413533 20936 20955 ATCCAGTTCACCATTGGAGA 48 166 413534 21441 21460 TTTTCCAGAAGAGACTCTTC 31 167 413535 21785 21804 GTCACATTTAAAATTTCCAA 41 168 413536 23422 23441 TTAATATACTGCAGAGAACC 37 169 413537 25893 25912 AGAAATATCCCCAGACAGAG 16 170

Example 2 Dose-Dependent Antisense Inhibition of Human Factor 11 in HepG2 Cells

Twelve gapmers, exhibiting over 84 percent or greater in vitro inhibition of human Factor 11, were tested at various doses in HepG2 cells. Cells were plated at a density of 10,000 cells per well and transfected using lipofectin reagent with 9.375 nM, 18.75 nM, 37.5 nM, 75 nM, and 150 nM concentrations of antisense oligonucleotide, as specified in Table 3. After a treatment period of approximately 16 hours, RNA was isolated from the cells and Factor 11 mRNA levels were measured by quantitative real-time PCR. Human Factor 11 primer probe set RTS 2966 (forward sequence: CAGCCTGGAGCATCGTAACA, incorporated herein as SEQ ID NO: 3; reverse sequence: TTTATCGAGCTTCGTTATTCTGGTT, incorporated herein as SEQ ID NO: 4; probe sequence: TTGTCTACTGAAGCACACCCAAACAGGGAX, incorporated herein as SEQ ID NO: 5) was used to measure mRNA levels. Factor 11 mRNA levels were adjusted according to total RNA content, as measured by RIBOGREEN. Results are presented as percent inhibition of Factor 11, relative to untreated control cells. As illustrated in Table 3, Factor 11 mRNA levels were reduced in a dose-dependent manner in antisense oligonucleotide treated cells.

TABLE 3 Dose-dependent antisense inhibition of human Factor 11 in HepG2 cells 9.375 18.75 37.5 75 150 IC₅₀ SEQ ID nM nM nM nM nM (nM) No. 412203 29 15 61 77 82 33 31 412206 28 44 68 80 89 22 34 412212 28 45 59 73 88 25 40 412223 33 48 62 76 81 21 51 412224 24 45 57 70 81 28 52 412225 32 42 65 78 73 23 53 413467 2 35 49 61 47 43 100 413468 14 34 56 78 75 35 101 413469 24 33 53 70 84 33 102 413476 26 44 64 73 82 25 109 413481 22 38 56 67 83 32 114 413482 26 39 59 74 82 28 115

Example 3 Antisense Inhibition of Human Factor 11 in HepG2 Cells by Oligonucleotides Designed by Microwalk

Additional gapmers were designed based on the gapmers presented in Table 3. These gapmers were designed by creating gapmers shifted slightly upstream and downstream (i.e. “microwalk”) of the original gapmers from Table 3. Gapmers were also created with various motifs, e.g. 5-10-5 MOE, 3-14-3 MOE, and 2-13-5 MOE. These gapmers were tested in vitro. Cultured HepG2 cells at a density of 10,000 cells per well were transfected using lipofectin reagent with 75 nM antisense oligonucleotide. After a treatment period of approximately 24 hours, RNA was isolated from the cells and Factor 11 mRNA levels were measured by quantitative real-time PCR.

Factor 11 mRNA levels were adjusted according to total RNA content, as measured by RIBOGREEN. Results are presented as percent inhibition of Factor 11, relative to untreated control cells.

The in vitro inhibition data for the gapmers designed by microwalk were then compared with the in vitro inhibition data for the gapmers from Table 3, as indicated in Tables 4, 5, 6, 7, and 8. The oligonucleotides are displayed according to the region on the human mRNA (GENBANK Accession No. NM_(—)000128.3) to which they map.

The chimeric antisense oligonucleotides in Table 4 were designed as 5-10-5 MOE, 3-14-3 MOE, and 2-13-5 MOE gapmers. The first listed gapmers in Table 4 are the original gapmers (see Table 3) from which the remaining gapmers were designed via microwalk and are designated by an asterisk. The 5-10-5 gapmers are 20 nucleotides in length, wherein the central gap segment is comprised of 10 2′-deoxynucleotides and is flanked on both sides (in the 5′ and 3′ directions) by wings comprising 5 nucleotides each. The 3-14-3 gapmers are 20 nucleotides in length, wherein the central gap segment is comprised of 14 2′-deoxynucleotides and is flanked on both sides (in the 5′and 3′ directions) by wings comprising 3 nucleotides each. The 2-13-5 gapmers are 20 nucleotides in length, wherein the central gap segment is comprised of 13 2′-deoxynucleotides. The central gap is flanked on the 5′ end with a wing comprising 2 nucleotides and on the 3′ end with a wing comprising 5 nucleotides. For each of the motifs (5-10-5, 3-14-3, and 2-13-5), each nucleotide in the 5′ wing segment and each nucleotide in the 3′ wing segment has a 2′-MOE modification. The internucleoside linkages throughout each gapmer are phosphorothioate (P═S) linkages. All cytidine residues throughout each gapmer are 5-methylcytidines. “Target start site” indicates the 5′-most nucleotide to which the gapmer is targeted. “Target stop site” indicates the 3′-most nucleotide to which the gapmer is targeted. Each gapmer listed in Table 4 is targeted to SEQ ID NO: 1 (GENBANK Accession No. NM_(—)000128.3).

As shown in Table 4, all of the 5-10-5 MOE gapmers, 3-14-3 MOE gapmers, and 2-13-5 MOE gapmers targeted to the target region beginning at target start site 656 and ending at the target stop site 704 (i.e. nucleobases 656-704) of SEQ ID NO: 1 exhibit at least 20% inhibition of Factor 11 mRNA. Many of the gapmers exhibit at least 60% inhibition. Several of the gapmers exhibit at least 80% inhibition, including ISIS numbers: 416806, 416809, 416811, 416814, 416821, 416825, 416826, 416827, 416828, 416868, 416869, 416878, 416879, 416881, 416883, 416890, 416891, 416892, 416893, 416894, 416895, 416896, 416945, 416946, 416969, 416970, 416971, 416972, 416973, 412203, 413467, 413468, and 413469. The following ISIS numbers exhibited at least 90% inhibition: 412203, 413467, 416825, 416826, 416827, 416868, 416878, 416879, 416892, 416893, 416895, 416896, 416945, 416972, and 416973. The following ISIS numbers exhibited at least 95% inhibition: 416878, 416892, 416895, and 416896.

TABLE 4 Inhibition of human Factor 11 mRNA levels by chimeric antisense oligonucleotides targeted to nucleobases 656 to 704 of SEQ ID NO: 1 (GENBANK Accession No. NM_000128.3) Target Target % SEQ ISIS No. Start Site Stop Site Sequence (5′ to 3′) inhibition Motif ID No. *412203 656 675 AATGTCTTTGTTGCAAGCGC 97 5-10-5 31 *413467 666 685 GGTCCACATAAATGTCTTTG 92 5-10-5 100 *413468 671 690 GTCTAGGTCCACATAAATGT 83 5-10-5 101 *413469 681 700 TGCCCTTCATGTCTAGGTCC 86 5-10-5 102 416868 656 675 AATGTCTTTGTTGCAAGCGC 93 3-14-3 31 416945 656 675 AATGTCTTTGTTGCAAGCGC 94 2-13-5 31 416806 657 676 AAATGTCTTTGTTGCAAGCG 86 5-10-5 171 416869 657 676 AAATGTCTTTGTTGCAAGCG 81 3-14-3 171 416946 657 676 AAATGTCTTTGTTGCAAGCG 86 2-13-5 171 416807 658 677 TAAATGTCTTTGTTGCAAGC 51 5-10-5 172 416870 658 677 TAAATGTCTTTGTTGCAAGC 76 3-14-3 172 416947 658 677 TAAATGTCTTTGTTGCAAGC 62 2-13-5 172 416808 659 678 ATAAATGTCTTTGTTGCAAG 55 5-10-5 173 416871 659 678 ATAAATGTCTTTGTTGCAAG 28 3-14-3 173 416948 659 678 ATAAATGTCTTTGTTGCAAG 62 2-13-5 173 416809 660 679 CATAAATGTCTTTGTTGCAA 86 5-10-5 174 416872 660 679 CATAAATGTCTTTGTTGCAA 20 3-14-3 174 416949 660 679 CATAAATGTCTTTGTTGCAA 64 2-13-5 174 416873 661 680 ACATAAATGTCTTTGTTGCA 51 3-14-3 99 416950 661 680 ACATAAATGTCTTTGTTGCA 71 2-13-5 99 416810 662 681 CACATAAATGTCTTTGTTGC 68 5-10-5 175 416874 662 681 CACATAAATGTCTTTGTTGC 49 3-14-3 175 416951 662 681 CACATAAATGTCTTTGTTGC 48 2-13-5 175 416811 663 682 CCACATAAATGTCTTTGTTG 84 5-10-5 176 416875 663 682 CCACATAAATGTCTTTGTTG 75 3-14-3 176 416952 663 682 CCACATAAATGTCTTTGTTG 51 2-13-5 176 416812 664 68 TCCACATAAATGTCTTTGTT 59 5-10-5 177 416876 664 683 TCCACATAAATGTCTTTGTT 37 3-14-3 177 416953 664 683 TCCACATAAATGTCTTTGTT 45 2-13-5 177 416813 665 684 GTCCACATAAATGTCTTTGT 70 5-10-5 178 416877 665 684 GTCCACATAAATGTCTTTGT 51 3-14-3 178 416954 665 684 GTCCACATAAATGTCTTTGT 61 2-13-5 178 416878 666 685 GGTCCACATAAATGTCTTTG 95 3-14-3 100 416955 666 685 GGTCCACATAAATGTCTTTG 75 2-13-5 100 416814 667 686 AGGTCCACATAAATGTCTTT 83 5-10-5 179 416879 667 686 AGGTCCACATAAATGTCTTT 92 3-14-3 179 416956 667 686 AGGTCCACATAAATGTCTTT 61 2-13-5 179 416815 668 687 TAGGTCCACATAAATGTCTT 63 5-10-5 180 416880 668 687 TAGGTCCACATAAATGTCTT 66 3-14-3 180 416957 668 687 TAGGTCCACATAAATGTCTT 59 2-13-5 180 416816 669 688 CTAGGTCCACATAAATGTCT 79 5-10-5 181 416881 669 688 CTAGGTCCACATAAATGTCT 81 3-14-3 181 416958 669 688 CTAGGTCCACATAAATGTCT 43 2-13-5 181 416817 670 689 TCTAGGTCCACATAAATGTC 74 5-10-5 182 416882 670 689 TCTAGGTCCACATAAATGTC 60 3-14-3 182 416959 670 689 TCTAGGTCCACATAAATGTC 25 2-13-5 182 416883 671 690 GTCTAGGTCCACATAAATGT 82 3-14-3 101 416960 671 690 GTCTAGGTCCACATAAATGT 60 2-13-5 101 416818 672 691 TGTCTAGGTCCACATAAATG 76 5-10-5 183 416884 672 691 TGTCTAGGTCCACATAAATG 69 3-14-3 183 416961 672 691 TGTCTAGGTCCACATAAATG 40 2-13-5 183 416819 673 692 ATGTCTAGGTCCACATAAAT 56 5-10-5 184 416885 673 692 ATGTCTAGGTCCACATAAAT 67 3-14-3 184 416962 673 692 ATGTCTAGGTCCACATAAAT 77 2-13-5 184 416820 674 693 CATGTCTAGGTCCACATAAA 77 5-10-5 185 416886 674 693 CATGTCTAGGTCCACATAAA 74 3-14-3 185 416963 674 693 CATGTCTAGGTCCACATAAA 48 2-13-5 185 416821 675 694 TCATGTCTAGGTCCACATAA 84 5-10-5 186 416964 675 694 TCATGTCTAGGTCCACATAA 69 2-13-5 186 412204 676 695 TTCATGTCTAGGTCCACATA 76 5-10-5 32 416888 676 695 TTCATGTCTAGGTCCACATA 76 3-14-3 32 416965 676 695 TTCATGTCTAGGTCCACATA 53 2-13-5 32 416822 677 696 CTTCATGTCTAGGTCCACAT 76 5-10-5 187 416889 677 696 CTTCATGTCTAGGTCCACAT 60 3-14-3 187 416966 677 696 CTTCATGTCTAGGTCCACAT 64 2-13-5 187 416823 678 697 CCTTCATGTCTAGGTCCACA 77 5-10-5 188 416890 678 697 CCTTCATGTCTAGGTCCACA 87 3-14-3 188 416967 678 697 CCTTCATGTCTAGGTCCACA 75 2-13-5 188 416824 679 698 CCCTTCATGTCTAGGTCCAC 64 5-10-5 189 416891 679 698 CCCTTCATGTCTAGGTCCAC 81 3-14-3 189 416968 679 698 CCCTTCATGTCTAGGTCCAC 73 2-13-5 189 416825 680 699 GCCCTTCATGTCTAGGTCCA 92 5-10-5 190 416892 680 699 GCCCTTCATGTCTAGGTCCA 100 3-14-3 190 416969 680 699 GCCCTTCATGTCTAGGTCCA 80 2-13-5 190 416893 681 700 TGCCCTTCATGTCTAGGTCC 90 3-14-3 102 416970 681 700 TGCCCTTCATGTCTAGGTCC 88 2-13-5 102 416826 682 701 ATGCCCTTCATGTCTAGGTC 94 5-10-5 191 416894 682 701 ATGCCCTTCATGTCTAGGTC 85 3-14-3 191 416971 682 701 ATGCCCTTCATGTCTAGGTC 83 2-13-5 191 416827 683 702 TATGCCCTTCATGTCTAGGT 93 5-10-5 192 416895 683 702 TATGCCCTTCATGTCTAGGT 95 3-14-3 192 416972 683 702 TATGCCCTTCATGTCTAGGT 90 2-13-5 192 416828 684 703 TTATGCCCTTCATGTCTAGG 87 5-10-5 193 416896 684 703 TTATGCCCTTCATGTCTAGG 95 3-14-3 193 416973 684 703 TTATGCCCTTCATGTCTAGG 92 2-13-5 193 416829 685 704 TTTATGCCCTTCATGTCTAG 72 5-10-5 194 416897 685 704 TTTATGCCCTTCATGTCTAG 66 3-14-3 194 416974 685 704 TTTATGCCCTTCATGTCTAG 73 2-13-5 194

The chimeric antisense oligonucleotides in Table 5 were designed as 5-10-5 MOE, 3-14-3 MOE, and 2-13-5 MOE gapmers. The first listed gapmer in Table 5 is the original gapmer (see Table 3) from which the remaining gapmers were designed via microwalk and is designated by an asterisk. The 5-10-5 gapmers are 20 nucleotides in length, wherein the central gap segment is comprised of 10 2′-deoxynucleotides and is flanked on both sides (in the 5′ and 3′ directions) by wings comprising 5 nucleotides each. The 3-14-3 gapmers are 20 nucleotides in length, wherein the central gap segment is comprised of 14 2′-deoxynucleotides and is flanked on both sides (in the 5′ and 3′ directions) by wings comprising 3 nucleotides each. The 2-13-5 gapmers are 20 nucleotides in length, wherein the central gap segment is comprised of 13 2′-deoxynucleotides. The central gap is flanked on the 5′ end with a wing comprising 2 nucleotides and on the 3′ end with a wing comprising 5 nucleotides. For each of the motifs (5-10-5, 3-14-3, and 2-13-5), each nucleotide in the 5′ wing segment and each nucleotide in the 3′ wing segment has a 2′-MOE modification. The internucleoside linkages throughout each gapmer are phosphorothioate (P═S) linkages. All cytidine residues throughout each gapmer are 5-methylcytidines. “Target start site” indicates the 5′-most nucleotide to which the gapmer is targeted. “Target stop site” indicates the 3′-most nucleotide to which the gapmer is targeted. Each gapmer listed in Table 5 is targeted to SEQ ID NO: 1 (GENBANK Accession No. NM_(—)000128.3).

As shown in Table 5, all of the 5-10-5 MOE gapmers, 3-14-3 MOE gapmers, and 2-13-5 MOE gapmers targeted to the target region beginning at target start site 738 and ending at the target stop site 762 (i.e. nucleobases 738-762) of SEQ ID NO: 1 exhibit at least 45% inhibition of Factor 11 mRNA. Most of the gapmers exhibit at least 60% inhibition. Several of the gapmers exhibit at least 80% inhibition, including ISIS numbers: 412206, 416830, 416831, 416898, 416899, 416900, 416903, 416975, 416976, 416977, and 416980. The following ISIS numbers exhibited at least 90% inhibition: 412206, 416831, and 416900.

TABLE 5 Inhibition of human Factor 11 mRNA levels by chimeric antisense oligonucleotides targeted to nucleobases 738 to 762 of SEQ ID NO: 1 (GENBANK Accession No. NM_000128.3) Target Target % SEQ ID ISIS No. Start Site Stop Site Sequence (5′ to 3′) inhibition Motif No. *412206 738 757 CCGTGCATCTTTCTTGGCAT 93 5-10-5 34 416898 738 757 CCGTGCATCTTTCTTGGCAT 88 3-14-3 34 416975 738 757 CCGTGCATCTTTCTTGGCAT 87 2-13-5 34 416830 739 758 TCCGTGCATCTTTCTTGGCA 81 5-10-5 195 416899 739 758 TCCGTGCATCTTTCTTGGCA 86 3-14-3 195 416976 739 758 TCCGTGCATCTTTCTTGGCA 83 2-13-5 195 416831 740 759 ATCCGTGCATCTTTCTTGGC 91 5-10-5 196 416900 740 759 ATCCGTGCATCTTTCTTGGC 90 3-14-3 196 416977 740 759 ATCCGTGCATCTTTCTTGGC 82 2-13-5 196 416832 741 760 CATCCGTGCATCTTTCTTGG 79 5-10-5 197 416901 741 760 CATCCGTGCATCTTTCTTGG 65 3-14-3 197 416978 741 760 CATCCGTGCATCTTTCTTGG 76 2-13-5 197 416833 742 761 TCATCCGTGCATCTTTCTTG 65 5-10-5 198 416902 742 761 TCATCCGTGCATCTTTCTTG 46 3-14-3 198 416979 742 761 TCATCCGTGCATCTTTCTTG 63 2-13-5 198 416834 743 762 GTCATCCGTGCATCTTTCTT 58 5-10-5 199 416903 743 762 GTCATCCGTGCATCTTTCTT 88 3-14-3 199 416980 743 762 GTCATCCGTGCATCTTTCTT 87 2-13-5 199

The chimeric antisense oligonucleotides in Table 6 were designed as 5-10-5 MOE, 3-14-3 MOE, and 2-13-5 MOE gapmers. The first listed gapmers in Table 6 are the original gapmers (see Table 3) from which the remaining gapmers were designed via microwalk and are designated by an asterisk. The 5-10-5 gapmers are 20 nucleotides in length, wherein the central gap segment is comprised of 10 2′-deoxynucleotides and is flanked on both sides (in the 5′ and 3′ directions) by wings comprising 5 nucleotides each. The 3-14-3 gapmers are 20 nucleotides in length, wherein the central gap segment is comprised of 14 2′-deoxynucleotides and is flanked on both sides (in the 5′ and 3′ directions) by wings comprising 3 nucleotides each. The 2-13-5 gapmers are 20 nucleotides in length, wherein the central gap segment is comprised of 13 2′-deoxynucleotides. The central gap is flanked on the 5′ end with a wing comprising 2 nucleotides and on the 3′ end with a wing comprising 5 nucleotides. For each of the motifs (5-10-5, 3-14-3, and 2-13-5), each nucleotide in the 5′ wing segment and each nucleotide in the 3′ wing segment has a 2′-MOE modification. The internucleoside linkages throughout each gapmer are phosphorothioate (P═S) linkages. All cytidine residues throughout each gapmer are 5-methylcytidines. “Target start site” indicates the 5′-most nucleotide to which the gapmer is targeted. “Target stop site” indicates the 3′-most nucleotide to which the gapmer is targeted. Each gapmer listed in Table 6 is targeted to SEQ ID NO: 1 (GENBANK Accession No. NM_(—)000128.3).

As shown in Table 6, all of the 5-10-5 MOE gapmers, 3-14-3 MOE gapmers, and 2-13-5 MOE gapmers targeted to the target region beginning at target start site 1018 and ending at the target stop site 1042 (i.e. nucleobases 1018-1042) of SEQ ID NO: 1 exhibit at least 80% inhibition of Factor 11 mRNA. The following ISIS numbers exhibited at least 90% inhibition: 413474, 416837, 416838, 416904, 416907, and 416908.

TABLE 6 Inhibition of human Factor 11 mRNA levels by chimeric antisense oligonucleotides targeted to nucleobases 1018 to 1042 of SEQ ID NO: 1 (GENBANK Accession No. NM_000128.3) Target Start Target Stop % SEQ ID ISIS No. Site Site Sequence (5′ to 3′) inhibition Motif No. *412212 1018 1037 CCGGGATGATGAGTGCAGAT 89 5-10-5 40 416904 1018 1037 CCGGGATGATGAGTGCAGAT 90 3-14-3 40 416981 1018 1037 CCGGGATGATGAGTGCAGAT 87 2-13-5 40 416835 1019 1038 ACCGGGATGATGAGTGCAGA 83 5-10-5 200 416905 1019 1038 ACCGGGATGATGAGTGCAGA 85 3-14-3 200 416982 1019 1038 ACCGGGATGATGAGTGCAGA 84 2-13-5 200 416836 1020 1039 AACCGGGATGATGAGTGCAG 89 5-10-5 201 416906 1020 1039 AACCGGGATGATGAGTGCAG 88 3-14-3 201 416983 1020 1039 AACCGGGATGATGAGTGCAG 86 2-13-5 201 416837 1021 1040 CAACCGGGATGATGAGTGCA 90 5-10-5 202 416907 1021 1040 CAACCGGGATGATGAGTGCA 90 3-14-3 202 416984 1021 1040 CAACCGGGATGATGAGTGCA 89 2-13-5 202 416838 1022 1041 GCAACCGGGATGATGAGTGC 94 5-10-5 203 416908 1022 1041 GCAACCGGGATGATGAGTGC 98 3-14-3 203 416985 1022 1041 GCAACCGGGATGATGAGTGC 88 2-13-5 203 413474 1023 1042 AGCAACCGGGATGATGAGTG 93 5-10-5 107

The chimeric antisense oligonucleotides in Table 7 were designed as 5-10-5 MOE, 3-14-3 MOE, and 2-13-5 MOE gapmers. The first listed gapmer in Table 7 is the original gapmer (see Table 3) from which the remaining gapmers were designed via microwalk and is designated by an asterisk. The 5-10-5 gapmers are 20 nucleotides in length, wherein the central gap segment is comprised of 10 2′-deoxynucleotides and is flanked on both sides (in the 5′ and 3′ directions) by wings comprising 5 nucleotides each. The 3-14-3 gapmers are 20 nucleotides in length, wherein the central gap segment is comprised of 14 2′-deoxynucleotides and is flanked on both sides (in the 5′ and 3′ directions) by wings comprising 3 nucleotides each. The 2-13-5 gapmers are 20 nucleotides in length, wherein the central gap segment is comprised of 13 2′-deoxynucleotides. The central gap is flanked on the 5′ end with a wing comprising 2 nucleotides and on the 3′ end with a wing comprising 5 nucleotides. For each of the motifs (5-10-5, 3-14-3, and 2-13-5), each nucleotide in the 5′ wing segment and each nucleotide in the 3′ wing segment has a 2′-MOE modification. The internucleoside linkages throughout each gapmer are phosphorothioate (P═S) linkages. All cytidine residues throughout each gapmer are 5-methylcytidines. “Target start site” indicates the 5′-most nucleotide to which the gapmer is targeted. “Target stop site” indicates the 3′-most nucleotide to which the gapmer is targeted. Each gapmer listed in Table 7 is targeted to SEQ ID NO: 1 (GENBANK Accession No. NM_(—)000128.3).

As shown in Table 7, all of the 5-10-5 MOE gapmers, 3-14-3 MOE gapmers, and 2-13-5 MOE gapmers targeted to the target region beginning at target start site 1062 and ending at the target stop site 1091 (i.e. nucleobases 1062-1091) of SEQ ID NO: 1 exhibit at least 20% inhibition of Factor 11 mRNA. Many of the gapmers exhibit at least 50% inhibition, including: 412215, 413476, 413476, 416839, 416840, 416841, 416842, 416843, 416844, 416845, 416846, 416847, 416909, 416910, 416911, 416912, 416913, 416914, 416915, 416916, 416917, 416918, 416986, 416987, 416988, 416989, 416990, 416991, 416992, 416993, 416994, 416995. The following ISIS numbers exhibited at least 80% inhibition: 412215, 413476, 413476, 416839, 416840, 416841, 416842, 416843, 416844, 416845, 416910, 416911, 416912, 416913, 416914, 416916, 416917, 416986, 416987, 416989, 416991, 416992, 416993, and 416994. The following ISIS numbers exhibited at least 90% inhibition: 413476, 413476, 416842, 416844, 416910, 416911, 416912, 416913, 416916, 416917, and 416993.

TABLE 7 Inhibition of human Factor 11 mRNA levels by chimeric antisense oligonucleotides targeted to nucleobases 1062 to 1091 of SEQ ID NO: 1 (GENBANK Accession No. NM_000128.3) ISIS Target Target % SEQ No. Start Site Stop Site Sequence (5′ to 3′) inhibition Motif ID No. *413476 1067 1086 TTGAGATTCTTTGGGCCATT 93 5-10-5 109 412215 1062 1081 ATTCTTTGGGCCATTCCTGG 82 5-10-5 43 416909 1062 1081 ATTCTTTGGGCCATTCCTGG 78 3-14-3 43 416986 1062 1081 ATTCTTTGGGCCATTCCTGG 88 2-13-5 43 416839 1063 1082 GATTCTTTGGGCCATTCCTG 89 5-10-5 204 416910 1063 1082 GATTCTTTGGGCCATTCCTG 90 3-14-3 204 416987 1063 1082 GATTCTTTGGGCCATTCCTG 80 2-13-5 204 416840 1064 1083 AGATTCTTTGGGCCATTCCT 85 5-10-5 205 416911 1064 1083 AGATTCTTTGGGCCATTCCT 90 3-14-3 205 416988 1064 1083 AGATTCTTTGGGCCATTCCT 76 2-13-5 205 416841 1065 1084 GAGATTCTTTGGGCCATTCC 87 5-10-5 206 416912 1065 1084 GAGATTCTTTGGGCCATTCC 92 3-14-3 206 416989 1065 1084 GAGATTCTTTGGGCCATTCC 88 2-13-5 206 416842 1066 1085 TGAGATTCTTTGGGCCATTC 94 5-10-5 207 416913 1066 1085 TGAGATTCTTTGGGCCATTC 93 3-14-3 207 416990 1066 1085 TGAGATTCTTTGGGCCATTC 76 2-13-5 207 413476 1067 1086 TTGAGATTCTTTGGGCCATT 93 5-10-5 109 416914 1067 1086 TTGAGATTCTTTGGGCCATT 87 3-14-3 109 416991 1067 1086 TTGAGATTCTTTGGGCCATT 87 2-13-5 109 416843 1068 1087 TTTGAGATTCTTTGGGCCAT 89 5-10-5 208 416915 1068 1087 TTTGAGATTCTTTGGGCCAT 79 3-14-3 208 416992 1068 1087 TTTGAGATTCTTTGGGCCAT 84 2-13-5 208 416844 1069 1088 CTTTGAGATTCTTTGGGCCA 90 5-10-5 209 416916 1069 1088 CTTTGAGATTCTTTGGGCCA 91 3-14-3 209 416993 1069 1088 CTTTGAGATTCTTTGGGCCA 91 2-13-5 209 416845 1070 1089 TCTTTGAGATTCTTTGGGCC 86 5-10-5 210 416917 1070 1089 TCTTTGAGATTCTTTGGGCC 92 3-14-3 210 416994 1070 1089 TCTTTGAGATTCTTTGGGCC 83 2-13-5 210 416846 1071 1090 TTCTTTGAGATTCTTTGGGC 72 5-10-5 211 416918 1071 1090 TTCTTTGAGATTCTTTGGGC 63 3-14-3 211 416995 1071 1090 TTCTTTGAGATTCTTTGGGC 64 2-13-5 211 416847 1072 1091 TTTCTTTGAGATTCTTTGGG 50 5-10-5 212 416919 1072 1091 TTTCTTTGAGATTCTTTGGG 27 3-14-3 212 416996 1072 1091 TTTCTTTGAGATTCTTTGGG 22 2-13-5 212

The chimeric antisense oligonucleotides in Table 8 were designed as 5-10-5 MOE, 3-14-3 MOE, and 2-13-5 MOE gapmers. The first listed gapmers in Table 8 are the original gapmers (see Table 3) from which the remaining gapmers were designed via microwalk and are designated by an asterisk. The 5-10-5 gapmers are 20 nucleotides in length, wherein the central gap segment is comprised of 10 2′-deoxynucleotides and is flanked on both sides (in the 5′ and 3′ directions) by wings comprising 5 nucleotides each. The 3-14-3 gapmers are 20 nucleotides in length, wherein the central gap segment is comprised of 14 2′-deoxynucleotides and is flanked on both sides (in the 5′ and 3′ directions) by wings comprising 3 nucleotides each. The 2-13-5 gapmers are 20 nucleotides in length, wherein the central gap segment is comprised of 13 2′-deoxynucleotides. The central gap is flanked on the 5′ end with a wing comprising 2 nucleotides and on the 3′ end with a wing comprising 5 nucleotides. For each of the motifs (5-10-5, 3-14-3, and 2-13-5), each nucleotide in the 5′ wing segment and each nucleotide in the 3′ wing segment has a 2′-MOE modification. The internucleoside linkages throughout each gapmer are phosphorothioate (P═S) linkages. All cytidine residues throughout each gapmer are 5-methylcytidines. “Target start site” indicates the 5′-most nucleotide to which the gapmer is targeted. “Target stop site” indicates the 3′-most nucleotide to which the gapmer is targeted. Each gapmer listed in Table 8 is targeted to SEQ ID NO: 1 (GENBANK Accession No. NM_(—)000128.3).

As shown in Table 8, all of the 5-10-5 MOE gapmers, 3-14-3 MOE gapmers, and 2-13-5 MOE gapmers targeted to the target region beginning at target start site 1275 and ending at the target stop site 1318 (i.e. nucleobases 1275-1318) of SEQ ID NO: 1 exhibit at least 70% inhibition of Factor 11 mRNA. Many of the gapmers exhibit at least 80% inhibition, including: 412223, 412224, 412225, 413482, 416848, 416849, 416850, 416851, 416852, 416853, 416854, 416855, 416856, 416857, 416858, 416859, 416860, 416861, 416862, 416863, 416864, 416865, 416866, 416867, 416920, 416921, 416922, 416923, 416924, 416925, 416926, 416927, 416928, 416929, 416930, 416931, 416932, 416933, 416934, 416935, 416936, 416937, 416938, 416939, 416940, 416941, 416942, 416943, 416944, 416997, 416998, 416999, 417000, 417001, 417002, 417003, 417004, 417006, 417007, 417008, 417009, 417010, 417011, 417013, 417014, 417015, 417016, 417017, 417018, 417019, and 417020. The following ISIS numbers exhibited at least 90% inhibition: 412224, 416850, 416853, 416856, 416857, 416858, 416861, 416862, 416864, 416922, 416923, 416924, 416925, 416926, 416928, 416931, 416932, 416933, 416934, 416935, 416937, 416938, 416940, 416941, 416943, 416999, 417002, 416854, and 416859.

TABLE 8 Inhibition of human Factor 11 mRNA levels by chimeric antisense oligonucleotides targeted to nucleobases 1275 to 1318 of SEQ ID NO: 1 (GENBANK Accession No. NM_000128.3) ISIS Target Target % SEQ No. Start Site Stop Site Sequence (5′ to 3′) inhibition Motif ID No. *412223 1275 1294 ACAGTTTCTGGCAGGCCTCG 85 5-10-5 51 *412224 1285 1304 GCATTGGTGCACAGTTTCTG 93 5-10-5 52 *413482 1290 1309 GGACGGCATTGGTGCACAGT 89 5-10-5 115 *412225 1295 1314 GCAGCGGACGGCATTGGTGC 86 5-10-5 53 416920 1275 1294 ACAGTTTCTGGCAGGCCTCG 88 3-14-3 51 416997 1275 1294 ACAGTTTCTGGCAGGCCTCG 84 2-13-5 51 416848 1276 1295 CACAGTTTCTGGCAGGCCTC 86 5-10-5 213 416921 1276 1295 CACAGTTTCTGGCAGGCCTC 88 3-14-3 213 416998 1276 1295 CACAGTTTCTGGCAGGCCTC 88 2-13-5 213 416849 1277 1296 GCACAGTTTCTGGCAGGCCT 88 5-10-5 214 416922 1277 1294 GCACAGTTTCTGGCAGGCCT 94 3-14-3 214 416999 1277 1296 GCACAGTTTCTGGCAGGCCT 92 2-13-5 214 416850 1278 1297 TGCACAGTTTCTGGCAGGCC 93 5-10-5 215 416923 1278 1297 TGCACAGTTTCTGGCAGGCC 96 3-14-3 215 417000 1278 1297 TGCACAGTTTCTGGCAGGCC 89 2-13-5 215 416851 1279 1298 GTGCACAGTTTCTGGCAGGC 88 5-10-5 216 416924 1279 1298 GTGCACAGTTTCTGGCAGGC 96 3-14-3 216 417001 1279 1298 GTGCACAGTTTCTGGCAGGC 83 2-13-5 216 416925 1280 1299 GGTGCACAGTTTCTGGCAGG 98 3-14-3 114 417002 1280 1299 GGTGCACAGTTTCTGGCAGG 92 2-13-5 114 416852 1281 1300 TGGTGCACAGTTTCTGGCAG 84 5-10-5 217 416926 1281 1300 TGGTGCACAGTTTCTGGCAG 93 3-14-3 217 417003 1281 1300 TGGTGCACAGTTTCTGGCAG 89 2-13-5 217 416853 1282 1301 TTGGTGCACAGTTTCTGGCA 91 5-10-5 218 416927 1282 1301 TTGGTGCACAGTTTCTGGCA 87 3-14-3 218 417004 1282 1301 TTGGTGCACAGTTTCTGGCA 86 2-13-5 218 416854 1283 1302 ATTGGTGCACAGTTTCTGGC 90 5-10-5 219 416928 1283 1302 ATTGGTGCACAGTTTCTGGC 91 3-14-3 219 417005 1283 1302 ATTGGTGCACAGTTTCTGGC 79 2-13-5 219 416855 1284 1303 CATTGGTGCACAGTTTCTGG 87 5-10-5 220 416929 1284 1303 CATTGGTGCACAGTTTCTGG 83 3-14-3 220 417006 1284 1303 CATTGGTGCACAGTTTCTGG 81 2-13-5 220 416930 1285 1304 GCATTGGTGCACAGTTTCTG 87 3-14-3 52 417007 1285 1304 GCATTGGTGCACAGTTTCTG 82 2-13-5 52 416856 1286 1305 GGCATTGGTGCACAGTTTCT 95 5-10-5 221 416931 1286 1305 GGCATTGGTGCACAGTTTCT 96 3-14-3 221 417008 1286 1305 GGCATTGGTGCACAGTTTCT 82 2-13-5 221 416857 1287 1306 CGGCATTGGTGCACAGTTTC 92 5-10-5 222 416932 1287 1306 CGGCATTGGTGCACAGTTTC 92 3-14-3 222 417009 1287 1306 CGGCATTGGTGCACAGTTTC 85 2-13-5 222 416858 1288 1307 ACGGCATTGGTGCACAGTTT 93 5-10-5 223 416933 1288 1307 ACGGCATTGGTGCACAGTTT 92 3-14-3 223 417010 1288 1307 ACGGCATTGGTGCACAGTTT 81 2-13-5 223 416859 1289 1308 GACGGCATTGGTGCACAGTT 90 5-10-5 224 416934 1289 1308 GACGGCATTGGTGCACAGTT 90 3-14-3 224 417011 1289 1308 GACGGCATTGGTGCACAGTT 86 2-13-5 224 416935 1290 1309 GGACGGCATTGGTGCACAGT 92 3-14-3 115 417012 1290 1309 GGACGGCATTGGTGCACAGT 72 2-13-5 115 416860 1291 1310 CGGACGGCATTGGTGCACAG 88 5-10-5 225 416936 1291 1310 CGGACGGCATTGGTGCACAG 89 3-14-3 225 417013 1291 1310 CGGACGGCATTGGTGCACAG 86 2-13-5 225 416861 1292 1311 GCGGACGGCATTGGTGCACA 92 5-10-5 226 416937 1292 1311 GCGGACGGCATTGGTGCACA 93 3-14-3 226 417014 1292 1311 GCGGACGGCATTGGTGCACA 87 2-13-5 226 416862 1293 1312 AGCGGACGGCATTGGTGCAC 90 5-10-5 227 416938 1293 1312 AGCGGACGGCATTGGTGCAC 90 3-14-3 227 417015 1293 1312 AGCGGACGGCATTGGTGCAC 87 2-13-5 227 416863 1294 1313 CAGCGGACGGCATTGGTGCA 83 5-10-5 228 416939 1294 1313 CAGCGGACGGCATTGGTGCA 88 3-14-3 228 417016 1294 1313 CAGCGGACGGCATTGGTGCA 85 2-13-5 228 416940 1295 1314 GCAGCGGACGGCATTGGTGC 92 3-14-3 53 417017 1295 1314 GCAGCGGACGGCATTGGTGC 82 2-13-5 53 416864 1296 1315 GGCAGCGGACGGCATTGGTG 93 5-10-5 229 416941 1296 1315 GGCAGCGGACGGCATTGGTG 95 3-14-3 229 417018 1296 1315 GGCAGCGGACGGCATTGGTG 82 2-13-5 229 416865 1297 1316 TGGCAGCGGACGGCATTGGT 88 5-10-5 230 416942 1297 1316 TGGCAGCGGACGGCATTGGT 85 3-14-3 230 417019 1297 1316 TGGCAGCGGACGGCATTGGT 84 2-13-5 230 416866 1298 1317 CTGGCAGCGGACGGCATTGG 88 5-10-5 231 416943 1298 1317 CTGGCAGCGGACGGCATTGG 92 3-14-3 231 417020 1298 1317 CTGGCAGCGGACGGCATTGG 84 2-13-5 231 416867 1299 1318 ACTGGCAGCGGACGGCATTG 83 5-10-5 232 416944 1299 1318 ACTGGCAGCGGACGGCATTG 83 3-14-3 232 417021 1299 1318 ACTGGCAGCGGACGGCATTG 74 2-13-5 232

Example 4 Dose-Dependent Antisense Inhibition of Human Factor 11 in HepG2 Cells

Gapmers from Example 3 (see Tables 4, 5, 6, 7, and 8), exhibiting in vitro inhibition of human Factor 11, were tested at various doses in HepG2 cells. Cells were plated at a density of 10,000 cells per well and transfected using lipofectin reagent with 9.375 nM, 18.75 nM, 37.5 nM and 75 nM concentrations of antisense oligonucleotide, as specified in Table 9. After a treatment period of approximately 16 hours, RNA was isolated from the cells and Factor 11 mRNA levels were measured by quantitative real-time PCR. Human Factor 11 primer probe set RTS 2966 was used to measure mRNA levels. Factor 11 mRNA levels were adjusted according to total RNA content, as measured by RIBOGREEN. Results are presented as percent inhibition of Factor 11, relative to untreated control cells. As illustrated in Table 9, Factor 11 mRNA levels were reduced in a dose-dependent manner in antisense oligonucleotide treated cells.

TABLE 9 Dose-dependent antisense inhibition of human Factor 11 in HepG2 cells via transfection of oligonucleotides with lipofectin 9.375 18.75 37.5 75 IC₅₀ SEQ ID nM nM nM nM Motif (nM) No. 412203 33 40 62 74 5-10-5 24 31 412206 24 47 69 86 5-10-5 21 34 413467 35 51 62 69 5-10-5 20 100 413474 29 44 57 67 5-10-5 28 107 413476 24 58 62 77 5-10-5 21 109 416825 23 52 73 92 5-10-5 20 190 416826 8 36 58 84 5-10-5 29 191 416827 31 42 62 77 5-10-5 23 192 416838 31 51 64 86 5-10-5 19 203 416842 18 33 62 71 5-10-5 31 207 416850 4 30 67 84 5-10-5 29 215 416856 21 45 58 74 5-10-5 27 221 416858 0 28 54 82 5-10-5 33 223 416864 18 43 62 78 5-10-5 26 229 416878 22 34 60 82 5-10-5 27 100 416892 16 50 70 85 3-14-3 23 190 416895 39 57 66 71 3-14-3 15 192 416896 22 39 57 81 3-14-3 27 193 416908 36 57 67 76 3-14-3 16 203 416922 14 25 49 75 3-14-3 36 214 416923 36 47 60 67 3-14-3 23 215 416924 25 38 56 59 3-14-3 36 216 416925 13 38 59 75 3-14-3 30 114 416926 31 43 63 82 3-14-3 22 217 416931 44 39 57 71 3-14-3 22 221 416941 33 54 63 78 3-14-3 19 229 416945 34 45 62 65 2-13-5 24 31 416969 17 39 61 76 2-13-5 28 190 416972 32 40 60 69 2-13-5 26 192 416973 60 75 85 87 2-13-5 3 193 416984 26 50 62 81 2-13-5 22 202 416985 17 30 47 57 2-13-5 49 203 416989 18 41 62 83 2-13-5 26 206 416993 15 37 50 68 2-13-5 36 209 416999 24 37 55 73 2-13-5 30 214 417000 35 47 58 70 2-13-5 23 215 417002 35 52 67 70 2-13-5 19 114 417003 26 44 60 56 2-13-5 33 217

The gapmers were also transfected via electroporation and their dose dependent inhibition of human Factor 11 mRNA was measured. Cells were plated at a density of 20,000 cells per well and transfected via electroporation with 0.7 μM, 2.2 μM, 6.7 μM, and 20 μM concentrations of antisense oligonucleotide, as specified in Table 10. After a treatment period of approximately 16 hours, RNA was isolated from the cells and Factor 11 mRNA levels were measured by quantitative real-time PCR. Human Factor 11 primer probe set RTS 2966 was used to measure mRNA levels. Factor 11 mRNA levels were adjusted according to total RNA content, as measured by RIBOGREEN. Results are presented as percent inhibition of Factor 11, relative to untreated control cells. As illustrated in Table 10, Factor 11 mRNA levels were reduced in a dose-dependent manner in antisense oligonucleotide treated cells.

TABLE 10 Dose-dependent antisense inhibition of human Factor 11 in HepG2 cells via transfection of oligonucleotides with electroporation 0.7 2.2 6.7 20 IC₅₀ SEQ ID μM μM μM μM (μM) No. 412203 11 60 70 91 2.7 31 412206 22 39 81 94 2.7 34 413467 5 31 65 89 4.2 100 413474 0 5 52 81 6.9 107 413476 40 69 88 93 0.9 109 416825 27 74 92 98 1.3 190 416826 2 47 86 82 3.2 191 416827 37 68 87 92 1.1 192 416838 5 30 55 83 5.1 203 416842 0 10 66 92 5.0 207 416850 14 25 81 91 3.4 215 416856 0 29 47 93 5.1 221 416858 5 20 56 86 5.3 223 416864 32 65 78 90 1.4 229 416878 1 26 75 85 4.3 100 416892 14 52 82 92 2.5 190 416895 0 62 70 91 3.0 192 416896 12 35 81 89 3.2 193 416908 7 58 74 89 2.8 203 416922 35 51 77 91 1.7 214 416923 15 30 60 90 4.0 215 416924 22 40 63 70 4.1 216 416925 0 40 76 80 3.9 114 416926 47 71 91 94 0.6 217 416931 7 24 60 82 5.1 221 416941 16 38 79 89 3.0 229 416945 48 70 81 88 0.6 31 416969 25 34 86 92 2.5 190 416972 25 30 48 88 4.3 192 416973 20 48 86 93 2.3 193 416984 43 54 88 90 1.1 202 416985 12 48 45 69 5.8 203 416989 32 65 88 94 1.3 206 416993 22 48 87 92 2.2 209 416999 20 42 77 88 2.8 214 417000 46 73 76 89 0.6 215 417002 32 38 82 91 2.2 114 417003 0 34 75 89 3.9 217

Example 5 Selection and Confirmation of Effective Dose-Dependent Antisense Inhibition of Human Factor 11 in HepG2 Cells

Gapmers exhibiting significant dose-dependent inhibition of human Factor 11 in Example 4 were selected and tested at various doses in HepG2 cells. Cells were plated at a density of 10,000 cells per well and transfected using lipofectin reagent with 2.34 nM, 4.69 nM, 9.375 nM, 18.75 nM, 37.5 nM, and 75 nM concentrations of antisense oligonucleotide, as specified in Table 11. After a treatment period of approximately 16 hours, RNA was isolated from the cells and human Factor 11 mRNA levels were measured by quantitative real-time PCR. Human Factor 11 primer probe set RTS 2966 was used to measure mRNA levels. Factor 11 mRNA levels were adjusted according to total RNA content, as measured by MBOGREEN. Results are presented as percent inhibition of human Factor 11, relative to untreated control cells. As illustrated in Table 11, Factor 11 mRNA levels were reduced in a dose-dependent manner in antisense oligonucleotide treated cells compared to the control.

TABLE 11 Dose-dependent antisense inhibition of human Factor 11 in HepG2 cells via transfection of oligonucleotides with lipofectin 2.34 4.69 9.375 18.75 37.5 75 IC50 SEQ ID nM nM nM nM nM nM Motif (nM) No. 416825 4 22 39 57 79 89 5-10-5 13 190 416826 15 22 32 54 76 90 5-10-5 15 191 416838 21 37 50 63 74 83 5-10-5 10 203 416850 24 31 49 55 70 77 5-10-5 13 215 416858 11 35 46 61 75 77 5-10-5 11 223 416864 13 34 42 65 68 80 5-10-5 15 229 416892 14 34 49 70 84 93 3-14-3 9 190 416925 24 34 45 56 67 72 3-14-3 13 114 416999 10 26 42 62 72 80 2-13-5 14 214 417002 17 26 49 61 81 84 2-13-5 12 114 417003 6 29 48 64 73 82 2-13-5 11 217

The gapmers were also transfected via electroporation and their dose dependent inhibition of human Factor 11 mRNA was measured. Cells were plated at a density of 20,000 cells per well and transfected via electroporation with 625 nM, 1250 nM, 2500 nM, 5,000 nM, 10,000 nM, and 20,000 nM concentrations of antisense oligonucleotide, as specified in Table 12. After a treatment period of approximately 16 hours, RNA was isolated from the cells and human Factor 11 mRNA levels were measured by quantitative real-time PCR. Human Factor 11 primer probe set RTS 2966 was used to measure mRNA levels. Factor 11 mRNA levels were adjusted according to total RNA content, as measured by RIBOGREEN. Results are presented as percent inhibition of human Factor 11, relative to untreated control cells. As illustrated in Table 12, Factor 11 mRNA levels were reduced in a dose-dependent manner in antisense oligonucleotide treated cells compared to the control.

TABLE 12 Dose-dependent antisense inhibition of human Factor 11 in HepG2 cells via transfection of oligonucleotides with electroporation 625 1250 2500 5000 10000 20000 IC50 SEQ ID nM nM nM nM nM nM (μM) No. 416825 69 84 91 94 96 97 19 190 416826 67 82 89 92 95 97 33 191 416838 66 79 87 90 93 96 43 203 416850 69 80 87 90 93 96 25 215 416858 65 77 87 89 93 93 44 223 416864 45 74 84 87 92 94 338 229 416892 66 86 96 97 100 100 31 190 416925 64 80 88 91 95 96 51 114 416999 61 82 89 94 94 97 67 214 417002 59 72 86 90 94 96 156 114 417003 60 74 86 90 95 95 123 217

Example 6 Selection and Confirmation of Effective Dose-Dependent Antisense Inhibition of Human Factor 11 in Cyano Primary Hepatocytes

Gapmers from Example 4 exhibiting significant dose dependent in vitro inhibition of human Factor 11 were also tested at various doses in cyano primary hepatocytes. Cells were plated at a density of 35,000 cells per well and transfected via electroporation with 0.74 nM, 2.2 nM, 6.7 nM, 20 nM, 60 nM, and 180 nM concentrations of antisense oligonucleotide, as specified in Table 13. After a treatment period of approximately 16 hours, RNA was isolated from the cells and human Factor 11 mRNA levels were measured by quantitative real-time PCR. Human Factor 11 primer probe set RTS 2966 was used to measure mRNA levels. Factor 11 mRNA levels were adjusted according to total RNA content, as measured by RIBOGREEN. Results are presented as percent inhibition of human Factor 11, relative to untreated control cells. As illustrated in Table 13, Factor 11 mRNA levels were reduced in a dose-dependent manner in antisense oligonucleotide treated cells compared to the control.

TABLE 13 Dose-dependent antisense inhibition of human Factor 11 in cyano primary hepatocytes 0.74 2.2 6.7 20 60 180 IC₅₀ SEQ ID nM nM nM nM nM nM (μM) No. 416825 5 22 51 61 77 84 1.0 190 416826 13 24 34 67 69 71 1.3 191 416838 0 0 21 34 48 62 6.9 203 416850 2 20 24 65 69 67 1.6 215 416858 2 13 22 44 63 68 3.7 223 416864 0 1 15 23 47 64 7.7 229 416892 20 20 43 62 88 92 1.0 190 416925 0 9 1 48 55 76 4.4 114 416999 3 40 36 62 67 82 1.3 214 417002 32 16 28 38 55 71 4.0 114 417003 12 18 19 39 58 74 4.1 217

Example 7 Selection and Confirmation of Effective Dose-Dependent Antisense Inhibition of Human Factor 11 in HepB3 Cells by Gapmers

Gapmers exhibiting in vitro inhibition of human Factor 11 in Example 4 were tested at various doses in human HepB3 cells. Cells were plated at a density of 4,000 cells per well and transfected using lipofectin reagent with 2.3 nM, 4.7 nM, 9.4 nM, 18.75 nM, 37.5 nM, and 75 nM concentrations of antisense oligonucleotide, as specified in Table 14. After a treatment period of approximately 16 hours, RNA was isolated from the cells and human Factor 11 mRNA levels were measured by quantitative real-time PCR. Human Factor 11 primer probe set RTS 2966 was used to measure mRNA levels. Factor 11 mRNA levels were adjusted according to total RNA content, as measured by RIBOGREEN. Results are presented as percent inhibition of Factor 11, relative to untreated control cells. As illustrated in Table 14, Factor 11 mRNA levels were reduced in a dose-dependent manner in antisense oligonucleotide treated cells compared to the control.

TABLE 14 Dose-dependent antisense inhibition of human Factor 11 in HepB3 cells 2.3 4.7 9.4 18.75 37.5 75 IC₅₀ SEQ ID ISIS No. nM nM nM nM nM nM (nM) No. 416825 0 15 34 36 53 59 35 190 416826 16 28 38 55 64 66 16 191 416838 23 34 43 59 71 56 11 203 416850 22 32 43 56 75 60 13 215 416858 17 34 43 57 74 62 12 223 416864 24 37 42 66 76 63 9 229 416892 28 34 50 68 82 72 9 190 416925 26 33 45 59 72 60 12 114 416999 19 33 42 60 71 59 12 214 417002 24 30 46 57 71 65 13 114 417003 11 28 40 40 63 58 17 217

The gapmers were also transfected via electroporation and their dose dependent inhibition of human Factor 11 mRNA was measured. Cells were plated at a density of 20,000 cells per well and transfected via electroporation with 41.15 nM, 123.457 nM, 370.37 nM, 1111.11 nM, 3333.33 nM, and 10,000 nM concentrations of antisense oligonucleotide, as specified in Table 15. After a treatment period of approximately 16 hours, RNA was isolated from the cells and human Factor 11 mRNA levels were measured by quantitative real-time PCR. Human Factor 11 primer probe set RTS 2966 was used to measure mRNA levels. Factor 11 mRNA levels were adjusted according to total RNA content, as measured by RIBOGREEN. Results are presented as percent inhibition of human Factor 11, relative to untreated control cells. As illustrated in Table 15, Factor 11 mRNA levels were reduced in a dose-dependent manner in antisense oligonucleotide treated cells compared to the control.

TABLE 15 Dose-dependent antisense inhibition of human Factor 11 in HepB3 cells 41.15 123.457 370.37 1111.11 3333.33 10000 IC₅₀ SEQ ID nM nM nM nM nM nM (μM) No. 416825 32 40 48 75 90 92 0.16 190 416826 0 0 34 61 87 92 0.78 191 416838 12 9 28 40 77 88 1.20 203 416850 26 38 51 73 90 95 0.30 215 416858 23 45 52 64 87 92 0.30 223 416864 4 3 6 35 75 87 2.20 229 416892 9 12 28 65 89 98 0.61 190 416925 27 39 50 73 88 96 0.20 114 416999 31 45 62 78 94 97 0.16 214 417002 19 0 31 47 86 93 1.20 114 417003 31 0 15 43 84 92 1.50 217

Example 8 Antisense Inhibition of Murine Factor 11 in Primary Mouse Hepatocytes

Chimeric antisense oligonucleotides targeting murine Factor 11 were designed as 5-10-5 MOE gapmers targeting murine Factor 11 (GENBANK Accession No. NM_(—)028066.1, incorporated herein as SEQ ID NO: 6). The gapmers are 20 nucleotides in length, wherein the central gap segment is comprised of 10 2′-deoxynucleotides and is flanked on both sides (in the 5′ and 3′ directions) by wings comprising 5 nucleotides each. Each nucleotide in each wing segment has a 2′-MOE modification. The internucleoside linkages throughout each gaper are phosphorothioate (P═S) linkages. All cytidine residues throughout each gapmer are 5-methylcytidines. The antisense oligonucleotides were evaluated for their ability to reduce murine Factor 11 mRNA in primary mouse hepatocytes.

Primary mouse hepatocytes were treated with 6.25 nM, 12.5 nM, 25 nM, 50 nM, 100 nM, and 200 nM of antisense oligonucleotides for a period of approximately 24 hours. RNA was isolated from the cells and murine Factor 11 mRNA levels were measured by quantitative real-time PCR. Murine Factor 11 primer probe set RTS 2898 (forward sequence ACATGACAGGCGCGATCTCT, incorporated herein as SEQ ID NO: 7; reverse sequence TCTAGGTTCACGTACACATCTTTGC, incorporated herein as SEQ ID NO: 8; probe sequence TTCCTTCAAGCAATGCCCTCAGCAATX, incorporated herein as SEQ ID NO: 9) was used to measure mRNA levels. Factor 11 mRNA levels were adjusted according to total RNA content as measured by RIBOGREEN. Several of the murine antisense oligonucleotides reduced Factor 11 mRNA levels in a dose-dependent manner.

Example 9 Cross-Reactive Antisense Inhibition of Murine Factor 11 in Primary Mouse Hepatocytes

Antisense oligonucleotides targeted to a murine factor 11 nucleic acid were tested for their effects on Factor 11 mRNA in vitro. Cultured primary mouse hepatocytes at a density of 10,000 cells per well were treated with 100 nM antisense oligonucleotide. After a treatment period of approximately 24 hours, RNA was isolated from the cells and mouse Factor 11 mRNA levels were measured by quantitative real-time PCR. Factor 11 mRNA levels were adjusted according to total RNA content, as measured by RIBOGREEN. Results are presented as percent inhibition of Factor 11, relative to untreated control cells.

The chimeric antisense oligonucleotides in Tables 16 were designed as 5-10-5 MOE gapmers. The gapmers are 20 nucleotides in length, wherein the central gap segment is comprised of 10 2′-deoxynucleotides and is flanked on both sides (in the 5′ and 3′ directions) by wings comprising 5 nucleotides each. Each nucleotide in the 5′ wing segment and each nucleotide in the 3′ wing segment has a 2′-MOE modification. The internucleoside linkages throughout each gapmer are phosphorothioate (P═S) linkages. All cytidine residues throughout each gapmer are 5-methylcytidines. “Mouse target start site” indicates the 5′-most nucleotide to which the gapmer is targeted. “Mouse target stop site” indicates the 3′-most nucleotide to which the gapmer is targeted. All the mouse oligonucleotides listed show cross-reactivity between the mouse Factor 11 mRNA (GENBANK Accession No. NM_(—)028066.1), incorporated herein as SEQ ID NO: 6 and the human Factor 11 mRNA (GENBANK Accession No. NM_(—)000128.3), incorporated herein as SEQ ID NO: 1. “Human Target Start Site” indicates the 5′-most nucleotide in the human mRNA (GENBANK Accession No. NM_(—)000128.3) to which the antisense oligonucleotide is targeted. “Human Target Stop Site” indicates the 3′-most nucleotide in the human mRNA (GENBANK Accession No. NM_(—)000128.3) to which the antisense oligonucleotide is targeted. “Number of mismatches” indicates the mismatches between the mouse oligonucleotide and the human mRNA sequence.

TABLE 16 Inhibition of mouse Factor 11 mRNA levels by chimeric antisense oligonucleotides having 5-10-5 MOE wings and deoxy gap targeted to SEQ ID NO: 1 and SEQ ID NO: 6 Mouse Mouse Human Human Target Target SEQ Target Target ISIS Start Stop % ID Start Stop No. of No Site Site Sequence (5′ to 3′) Inhibition No. Site Site mismatches 404050 379 398 TGCTTGAAGGAATATCCAGA 82 233 619 638 2 404054 448 467 TAGTTCATGCCCTTCATGTC 45 234 688 707 1 404055 453 472 TGTTATAGTTCATGCCCTTC 27 235 693 712 1 404066 686 705 AATGTCCCTGATACAAGCCA 37 236 926 945 1 404067 691 710 GGGAAAATGTCCCTGATACA 39 237 931 950 1 404083 1299 1318 TGTGCAGAGTCACCTGCCAT 47 238 1533 1552 2 404087 1466 1485 TTCTTGAACCCTGAAGAAAG 29 239 1709 1728 2 404089 1477 1496 TGAATTATCATTTCTTGAAC 6 240 1720 1739 2 404090 1483 1502 TGATCATGAATTATCATTTC 42 241 1726 1745 2

Example 10 In Vivo Antisense Inhibition of Murine Factor 11

Several antisense oligonucleotides targeted to murine Factor 11 mRNA (GENBANK Accession No. NM_(—)028066.1, incorporated herein as SEQ ID NO: 6) showing statistically significant dose-dependent inhibition were evaluated in vivo. BALB/c mice were treated with ISIS 404057 (TCCTGGCATTCTCGAGCATT, target start site 487, incorporated herein as SEQ ID NO: 10) and ISIS 404071 (TGGTAATCCACTTTCAGAGG, target start site 869, incorporated herein as SEQ ID NO: 11).

Treatment

BALB/c mice were injected with 5 mg/kg, 10 mg/kg, 25 mg/kg, or 50 mg/kg of ISIS 404057 or ISIS 404071 twice a week for 3 weeks. A control group of mice was injected with phosphate buffered saline (PBS) twice a week for 3 weeks. Mice were sacrificed 5 days after receiving the last dose. Whole liver was harvested for RNA analysis and plasma was collected for clotting analysis (PT and aPTT) and protein analysis.

RNA Analysis

RNA was extracted from liver tissue for real-time PCR analysis of Factor 11. As shown in Table 17, the antisense oligonucleotides achieved dose-dependent reduction of murine Factor 11 over the PBS control. Results are presented as percent inhibition of Factor 11, relative to control.

TABLE 17 Dose-dependent antisense inhibition of murine Factor 11 mRNA in BALB/c mice mg/kg % inhibition 404057 5 40 10 64 25 85 50 95 404071 5 72 10 82 25 93 50 96 PT and aPTT Assay

Prothrombin Time (PT) and Activated Partial Thromboplastin Time (aPTT) were measured using platelet poor plasma (PPP) from mice treated with ISIS 404057 and ISIS 404071. PT and aPTT values provided in Table 18 are reported as International Normalized Ratio (INR) values. INR values for PT and aPTT were determined by dividing the PT or aPTT value for each experimental group (i.e. 5 mg/kg, 10 mg/kg, 25 mg/kg, and 50 mg/kg treatment with ISIS 404057 or ISIS 404071) by the PT or aPTT for the PBS treated group. This ratio was then raised to the power of the International Sensitivity Index (ISI) of the tissue factor used. As shown in Table 18, PT was not significantly prolonged in mice treated with ISIS 404057 or ISIS 404071. However, aPTT was prolonged in a dose-dependent manner in mice treated with ISIS 404057 and ISIS 404071. These data suggest that antisense reduction of Factor 11 affects the contact activation pathway, but not the extrinsic pathway of blood coagulation.

TABLE 18 Effect of ISIS 404071 and 404057 on PT and aPTT in BALB/c mice Dose in mg/kg PT INR aPTT INR ISIS 404057 5 1.00 1.07 10 0.94 1.19 25 1.02 1.27 50 1.00 1.37 ISIS 404071 5 1.06 1.09 10 1.08 1.13 25 1.06 1.35 50 1.02 2.08

Protein Analysis

Factor 11 proenzyme from the plasma of mice treated with ISIS 404071, was measured using a F11 assay based on clotting time. Clotting times were determined in duplicate with a ST4 semi-automated coagulation instrument (Diagnostica Stago, N.J.). Thirty μl of citrated sample plasma diluted 1/20 in HEPES-NaCl buffer with BSA was incubated with 30 μl aPTT reagent (Platelet Factor 3 reagent plus particulate activator) and 30 μl of citrated plasma deficient of Factor 11 (human congential, George King Bio-Medical Inc.) at 37° C. to initiate clotting. Results were interpolated on a standard curve of serially diluted citrated control murine plasma.

As shown in Table 19, treatment with ISIS 404071 resulted in a significant dose-dependent reduction of Factor 11 protein. Results are presented as percent inhibition of Factor 11, relative to PBS control.

TABLE 19 Dose-dependent inhibition of murine Factor 11 protein by ISIS 404071 in BALB/c mice Dose in mg/kg % Inhibition 5 39 10 67 25 89 50 96

Example 11 In Vivo Effect of Antisense Inhibition of Murine Factor 11 in the FeCl₃ Induced Venous Thrombosis (VT) Model as Compared to Warfarin Treatment

ISIS 404071 and warfarin (COUMADIN) were evaluated in the FeCl₃ induced VT mouse model. Six groups of BALB/c mice were treated with 1.25 mg/kg, 2.5 mg/kg, 5 mg/kg, 10 mg/kg, 20 mg/kg, or 40 mg/kg of ISIS 404071, administered subcutaneously twice a week for 3 weeks. Two days after receiving the last dose of ISIS 404071, mice were anesthetized with 150 mg/kg ketamine mixed with 10 mg/kg xylazine administered by intraperitoneal injection. An additional 6 groups of BALB/c mice were treated with 0.5 mg/kg, 1 mg/kg, 2 mg/kg, 3 mg/kg, 4 mg/kg, and 5 mg/kg of warfarin, administered intraperioneally daily for 6 days. Four hours after the last dose of warfarin, mice were anesthetized with 150 mg/kg ketamine mixed with 10 mg/kg xylazine administered by intraperitoneal injection. Two control groups of BALB/c mice were treated with PBS, administered subcutaneously twice a week for 3 weeks. Two days after the last dose of PBS, mice in both groups were anesthetized with 150 mg/kg ketamine mixed with 10 mg/kg xylazine administered by intraperitoneal injection. Thrombus formation was induced with FeCl₃ in all groups of mice except the first control group.

In mice undergoing FeCl₃ treatment, thrombus formation was induced by applying a piece of filter paper (2×4 mm) pre-saturated with 10% FeCl₃ solution directly on the vena cava. After 3 minutes of exposure, the filter paper was removed. Thirty minutes after the filter paper application, a fixed length of the vein containing the thrombus was dissected out for platelet analysis. Liver was collected for RNA analysis.

RNA Analysis

RNA was extracted from liver tissue for real-time PCR analysis of Factor 11. Results are presented as percent inhibition of Factor 11, relative to PBS control. As shown in Table 20, treatment with ISIS 404071 resulted in significant dose-dependent reduction of Factor 11 mRNA in comparison to the PBS control. Conversely, treatment with warfarin did not result in significant reduction of Factor 11 as compared to the PBS control.

TABLE 20 Dose-dependent reduction of Factor 11 mRNA in the FeCl₃ induced venous thrombosis model Dose in Treatment mg/kg % inhibition Warfarin 0.5 0 1 0 2 1 3 5 4 8 5 11 ISIS 404071 1.25 0 2.5 8 5 62 10 78 20 92 40 96

Quantification of Platelet Composition

Real-time PCR quantification of platelet factor-4 (PF-4) was used to quantify platelets in the vena cava as a measure of thrombus formation. Results are presented as a percentage of PF-4 in ISIS 404071 or warfarin treated mice, as compared to the two PBS-treated control groups. As shown in Table 21, treatment with ISIS 404071 resulted in a dose-dependent reduction of PF-4 in comparison to the PBS control for dosages of 5 mg/kg and higher. Treatment with warfarin resulted in a reduction of PF-4 in comparison to the PBS control for dosages of 2 mg/kg and higher. Therefore, reduction of Factor 11 by the compounds provided herein is useful for inhibiting thrombus and clot formation.

TABLE 21 Analysis of thrombus formation by real-time PCR quantification of PF-4 in the FeCl₃ induced venous thrombosis model Dose in mg/kg PF-4 PBS − FeCl₃ 0 PBS + FeCl₃ 100 Warfarin 0.5 128 1 124 2 80 3 21 4 12 5 33 ISIS 404071 1.25 143 2.5 120 5 95 10 21 20 37 40 20

Example 12 In Vivo Effect of Antisense Inhibition of Murine Factor 11 Compared to Warfarin in a Tail Bleeding Assay Treatment

Tail-bleeding was measured to observe whether treatment with ISIS 404071 or warfarin causes internal hemorrhage in mice. ISIS 404071 and warfarin (COUMADIN) were evaluated in the tail bleeding assay. Six groups of BALB/c mice were treated with 1.25 mg/kg, 2.5 mg/kg, 5 mg/kg, 10 mg/kg, 20 mg/kg, or 40 mg/kg of ISIS 404071, administered subcutaneously twice a week for 3 weeks. An additional 6 groups of BALB/c mice were treated with 0.5 mg/kg, 1 mg/kg, 2 mg/kg, 3 mg/kg, 4 mg/kg, and 5 mg/kg of warfarin, administered intraperioneally daily for 6 days. A separate control group of BALB/c mice was treated with PBS, administered subcutaneously twice a week for 3 weeks.

Tail-Bleeding Assay

Two days after the final treatment of ISIS 404071, warfarin, or PBS, mice were placed in a tail bleeding chamber. Mice were anesthetized in the chamber with isoflurane and a small piece of tail (approximately 4 mm from the tip) was cut with sterile scissors. The tail cut was immediately placed in a 15 mL Falcon tube filled with approximately 10 mL of 0.9% NaCl buffer solution warmed to 37° C. The blood was collected over the course of 40 minutes. The saline filled tubes were weighed both before and after bleeding. The results are provided in Table 22.

Treatment with ISIS 404071 did not affect bleeding as compared to PBS treated mice. However, warfarin did increase bleeding in mice as compared to the PBS control. Increased doses of warfarin correlated positively with increased blood loss. These data suggest that the hemorrhagic potential of the compounds provided herein is low, especially in comparison to warfarin. These data taken with the results provided in example 11 suggest inhibition of Factor 11 with the compounds described herein are useful for providing antithrombotic activity without associated bleeding risk.

TABLE 22 Tail bleeding assay in the FeCl₃ induced venous thrombosis model Dose in Blood Treatment mg/kg (g) PBS 0 0.01 Warfarin 0.5 0.07 1 0.35 2 0.39 3 0.51 4 0.52 5 0.76 ISIS 404071 1.25 0.00 2.5 0.00 5 0.03 10 0.00 20 0.06 40 0.03

Example 13 In Vivo Effect of Antisense Inhibition of Murine Factor 11 Compared to Warfarin on PT and aPTT Treatment

PT and aPTT were measured using PPP from mice treated with ISIS 404071 or warfarin. Six groups of BALB/c mice were treated with 1.25 mg/kg, 2.5 mg/kg, 5 mg/kg, 10 mg/kg, 20 mg/kg, or 40 mg/kg of ISIS 404071, administered subcutaneously twice a week for 3 weeks. An additional 6 groups of BALB/c mice were treated with 0.5 mg/kg, 1 mg/kg, 2 mg/kg, 3 mg/kg, 4 mg/kg, and 5 mg/kg of warfarin, administered intraperioneally daily for 6 days. In a control group, BALB/c mice were treated with PBS, administered subcutaneously mice twice a week for 3 weeks.

Two days after the final dose was administered, PPP was collected and PT and aPTT assays were performed.

PT and aPTT Assay

PT and aPTT values provided in Table 16 are reported as International Normalized Ratio (INR) values. INR values for PT and aPTT were determined by dividing the PT or aPTT value for each experimental group (i.e. 5 mg/kg, 10 mg/kg, 25 mg/kg, and 50 mg/kg treatment with ISIS 404071) by the PT or aPTT for the PBS treated group. This ratio was then raised to the power of the International Sensitivity Index (ISI) of the tissue factor used. As shown in Table 23, PT in warfarin treated mice is significantly prolonged at every dosage. aPTT in warfarin treated mice was prolonged, particularly at dosages of 1 mg/kg and higher. ISIS 404071 did not significantly affect PT, but did prolong aPTT; however, not as significantly as in warfarin treated mice. These data suggest that ISIS 404071 affects the contact activation pathway, but not the extrinsic pathway of blood coagulation whereas warfarin affects both the contact activation pathway and the extrinsic pathway of blood coagulation.

TABLE 23 Effect of ISIS 404071 and warfarin on PT and aPTT in BALB/c mice Dose in Treatment mg/kg PT INR aPTT INR Warfarin 0.5 1.41 1.10 1 2.03 1.31 2 2.77 1.54 3 22.76 2.90 4 6.74 2.18 5 9.20 2.29 ISIS 404071 1.25 0.99 0.98 2.5 1.01 1.03 5 1.07 1.09 10 1.08 1.29 20 1.09 1.32 40 0.98 1.64

Example 14 In Vivo Effect of Antisense Inhibition of Murine Factor 11 in the FeCl₃ Induced Venous Thrombosis (VT) Model as Compared to Apixaban Treatment

ISIS 404071 and Apixaban were evaluated in the FeCl₃ induced VT mouse model. Six groups of BALB/c mice were treated with 1.25 mg/kg, 2.5 mg/kg, 5 mg/kg, 10 mg/kg, 20 mg/kg, or 40 mg/kg of ISIS 404071, administered subcutaneously twice a week for 3 weeks. Two days after receiving the last dose of ISIS 404071, mice were anesthetized with 150 mg/kg ketamine mixed with 10 mg/kg xylazine administered by intraperitoneal injection. An additional 6 groups of BALB/c mice were treated with 0.5 mg/kg, 1 mg/kg, 2 mg/kg, 3 mg/kg, 4 mg/kg, and 5 mg/kg of Apixaban, administered subcutaneously one time. Twenty minutes after receiving Apixaban, mice were anesthetized with 150 mg/kg ketamine mixed with 10 mg/kg xylazine administered by intraperitoneal injection. Two control groups of BALB/c mice were treated with PBS, administered subcutaneously twice a week for 3 weeks. Two days after the last dose of PBS, mice in both groups were anesthetized with 150 mg/kg ketamine mixed with 10 mg/kg xylazine administered by intraperitoneal injection. Thrombus formation was induced with FeCl₃ in all of the mice except the first control group.

In mice undergoing FeCl₃ treatment, thrombus formation was induced by applying a piece of filter paper (2×4 mm) pre-saturated with 10% FeCl₃ solution directly on the vena cava. After 3 minutes of exposure, the filter paper was removed. Thirty minutes after the filter paper application, a fixed length of the vein containing the thrombus was dissected out for platelet analysis. Liver was collected for RNA analysis.

RNA Analysis

RNA was extracted from liver tissue for real-time PCR analysis of Factor 11. Results are presented as percent inhibition of Factor 11, relative to PBS control. As shown in Table 24, treatment with ISIS 404071 resulted in significant dose-dependent reduction of Factor 11 mRNA in comparison to the PBS control. Conversely, treatment with Apixaban did not result in significant reduction of Factor 11 as compared to the PBS control.

TABLE 24 Dose-dependent reduction of Factor 11 mRNA in the FeCl₃ induced venous thrombosis model Dose in mg/kg % inhibition Apixaban 0.5 5 2 8 5 12 10 2 20 0 ISIS 404071 1.25 15 2.5 44 5 63 10 76 25 91 50 95

Quantification of Platelet Composition

Real-time PCR quantification of platelet factor-4 (PF-4) was used to quantify platelets in the vena cava as a measure of thrombus formation. As shown in Table 25, treatment with ISIS 404071 resulted in reduction of PF-4 in comparison to the PBS control. Treatment with Apixaban also resulted in reduction of PF-4, in comparison to the PBS control. Results are presented as a percentage of PF-4 in ISIS 404071 or Apixaban treated mice, as compared to the two PBS-treated control groups.

TABLE 25 Analysis of thrombus formation by real-time PCR quantification of PF-4 in the FeCl₃ induced venous thrombosis model Dose in Treatment mg/kg PF-4 PBS − FeCl₃ 0 PBS + FeCl₃ 100 Apixaban 0.5 67 2 46 5 15 10 5 20 26 ISIS 404071 1.25 42 2.5 87 5 60 10 28 25 14 50 4

Example 15 In Vivo Effect of Antisense Inhibition of Murine Factor 11 Compared to Apixaban in the Tail Bleeding Assay Treatment

Tail bleeding was measured to observe whether treatment with ISIS 404071 or warfarin causes internal hemorrhage in mice. ISIS 404071 and Apixaban were evaluated in the tail bleeding model. Six groups of BALB/c mice were treated with 1.25 mg/kg, 2.5 mg/kg, 5 mg/kg, 10 mg/kg, 20 mg/kg, or 40 mg/kg of ISIS 404071, administered subcutaneously twice a week for 3 weeks. An additional 6 groups of BALB/c mice were treated with 0.5 mg/kg, 1 mg/kg, 2 mg/kg, 3 mg/kg, 4 mg/kg, and 5 mg/kg of Apixaban, administered in a single subcutaneous dose. A separate control group of BALB/c mice was treated with PBS, administered subcutaneously twice a week for 3 weeks.

Tail-Bleeding Assay

Two days after the final treatment of ISIS 404071, Apixaban, or PBS, mice were placed in a tail bleeding chamber. Mice were anesthetized in the chamber and a small piece of tail (approximately 4 mm from the tip) was cut with sterile scissors. The cut tail was immediately placed in a 15 mL Falcon tube filled with approximately 10 mL of 0.9% NaCl buffer solution warmed to 37° C. The blood was collected over the course of 40 minutes. The saline filled tubes were weighed before and after bleeding.

As shown in Table 26, treatment with ISIS 404071 did not affect bleeding as compared to PBS treated mice. However, Apixaban did increase bleeding in mice as compared to the PBS control. Increased doses of Apixaban correlated positively with increased blood loss. These data suggest that the hemorrhagic potential of the compounds provided herein is low, especially in comparison to Apixaban. These data taken with the results provided in example 14 suggest inhibition of Factor 11 with the compounds described herein are useful for providing antithrombotic without associated bleeding risk.

TABLE 26 Tail bleeding assay in BABL/c mice mg/kg Blood (g) PBS 0 0.06 Apixaban 0.5 0.03 2 0.34 5 0.37 10 0.40 20 0.52 ISIS 404071 1.25 0.00 2.5 0.03 5 0.00 10 0.04 25 0.01 50 0.01

Example 16 Ex Vivo Effect of Antisense Inhibition of Murine Factor 11 in Combination with LOVENOX Treatment

Three groups of BALB/c mice were treated with 10 mg/kg, 20 mg/kg, or 40 mg/kg of ISIS 404071, administered subcutaneously twice a week for 3 weeks. A control mouse group was treated with PBS, administered twice a week for 3 weeks. Five days after the last dose, the mice were sacrificed and plasma was collected. The low-molecular-weight (LMW) heparin, LOVENOX, was administered to the plasma ex vivo at varying concentrations of 0 μg/ml, 2.5 μg/ml, 5.0 μg/ml, and 7.5 μg/ml. PT and aPTT were measured 20 minutes after LOVENOX was administered.

PT and aPTT Assay

As shown in Table 27, treatment with LOVENOX increases PT in a dose-dependent manner. Treatment with ISIS 404071 does not significantly increase PT. PT is not significantly affected by treatment with ISIS 404071. There is no evidence of a combinational effect on PT in ISIS 404071 and LOVENOX treated plasma.

TABLE 27 Effect of combination of ISIS 404071 and LOVENOX on PT INR in murine plasma ISIS 404071 LOVENOX (mg/ml) (mg/kg) 0 2.5 5.0 7.5 0 1.00 1.02 1.10 1.12 10 0.97 1.07 1.10 1.12 20 1.00 1.10 1.07 1.10 40 0.97 1.02 1.07 1.10

As shown in Table 28, treatment with LOVENOX increases aPTT in a dose-dependent manner. Treatment with ISIS 404071 also increases aPTT in a dose-dependent manner. Furthermore, the combined treatment of ISIS 404071 and LOVENOX appears to have a synergistic effect on aPTT.

TABLE 28 Effect of combination of ISIS 404071 and LOVENOX on aPTT INR in murine plasma ISIS 404071 LOVENOX (mg/ml) mg/kg 0 2.5 5.0 7.5 0 1.00 1.53 2.10 2.70 10 1.14 1.76 2.39 3.20 20 1.28 1.95 2.83 3.65 40 1.52 2.66 n.d. 4.78 n.d. = no data

Example 17 In Vivo Effect of Antisense Inhibition of Murine Factor 11 in Combination with LOVENOX in the FeCl₃ Induced Venous Thrombosis (VT) Model Treatment

The combination of ISIS 404071 and LOVENOX were evaluated in the FeCl₃ induced VT mouse model. Four groups of BALB/c mice were treated with 15 mg/kg, 30 mg/kg, 45 mg/kg, or 60 mg/kg of LOVENOX, administered subcutaneously once daily for 3 days. An additional 4 groups of BALB/c mice were treated with 20 mg/kg of ISIS 404071, administered subcutaneously twice weekly for 3 weeks. After the last dose of ISIS 404071, mice were treated with 15 mg/kg, 30 mg/kg, 45 mg/kg, or 60 mg/kg of LOVENOX, administered subcutaneously once daily for 3 days. Two control groups of BALB/c mice were treated with PBS, administered subcutaneously twice a week for 3 weeks. Thrombus formation was induced with FeCl₃ in all of the mice except the first control group. All mice were anesthetized with 150 mg/kg of ketamine mixed with 10 mg/kg of xylazine administered by intraperitoneal injection.

In mice undergoing FeCl₃ treatment, thrombus formation was induced by applying a piece of filter paper (2×4 mm) pre-saturated with 10% FeCl₃ solution directly on the vena cava. After 3 minutes of exposure, the filter paper was removed. Thirty minutes after the filter paper application, a fixed length of the vein containing the thrombus was dissected out for platelet analysis.

Quantification of Platelet Composition

Real-time PCR quantification of PF-4 was used to quantify platelets in the vena cava as a measure of thrombus formation. As shown in Table 29, treatment with LOVENOX resulted in a reduction of PF-4 in comparison to the PBS control. Treatment with LOVENOX in combination with ISIS 404071 resulted in a higher reduction of PF-4 in comparison to LOVENOX alone.

TABLE 29 Analysis of thrombus formation by real-time PCR quantification of PF-4 in the FeCl₃ induced venous thrombosis model Treatment mg/kg PF-4 PBS − FeCl₃ 0 PBS + FeCl₃ 100 LOVENOX 15 57 30 33 45 10 60 5 LOVENOX (+ISIS 15 0 404071) 30 0 45 11 60 5

Example 18 In Vivo Effect of Antisense Inhibition of Murine Factor 11 in Combination with LOVENOX on Bleeding Treatment

Tail-bleeding was measured to observe whether treatment with ISIS 404071 and LOVENOX causes internal hemorrhage in mice. ISIS 404071 was administered subcutaneously at a dosage of 20 mg/kg twice a week for 3 weeks to 4 groups of BALB/c mice, and LOVENOX was administered subcutaneously at varying dosages of 15 mg/kg, 30 mg/kg, 45 mg/kg, and 60 mg/kg once daily on the last three days of ISIS 404071 treatment. In a fifth group, ISIS 404071 was administered subcutaneously to BALB/c mice at a dosage of 20 mg/kg twice a week for 3 weeks. In a sixth group, PBS was administered subcutaneously twice a week for three weeks to BALB/c mice, as a control.

Tail-Bleeding Assay

Two days after receiving their final treatment, mice were placed in a tail bleeding chamber. Mice were anesthetized in the chamber with isoflurane and a small piece of tail (approximately 4 mm from the tip) was cut with sterile scissors. The cut tail was immediately placed in a 15 mL Falcon tube filled with approximately 10 mL of 0.9% NaCl buffer solution warmed to 37° C. The blood was collected over the course of 40 minutes. The saline filled tubes were weighed both before and after bleeding.

As shown in Table 30, LOVENOX increased bleeding in mice compared to the PBS treated mice. Increased doses of LOVENOX correlated positively with increased blood loss. ISIS 404071 combined with LOVENOX did not significant increase bleeding beyond the increased blood loss shown in LOVENOX only treated mice.

TABLE 30 Tail bleeding assay comparing LOVENOX and the combination of LOVENOX and ISIS 404071 Dose in Blood mg/kg (g) PBS 0.05 LOVENOX 15 0.11 30 0.20 45 0.27 60 0.47 LOVENOX (+ISIS 15 0.14 404071) 30 0.19 45 0.36 60 0.61

Example 19 In Vivo Effect of Antisense Inhibition of Murine Factor 11 in Combination with LOVENOX on PT and aPTT Treatment

PT and aPTT were measured using PPP from mice treated with ISIS 404071 in combination with LOVENOX. In the first cohort, ISIS 404071 was administered subcutaneously to BALB/c mice at a dosage of 25 mg/kg twice a week for 3 weeks. Plasma was collected from these mice 5 days after receiving the last dose of ISIS 404071. In the second cohort, LOVENOX was administered subcutaneously to BALB/c mice at a dosage of 20 mg/kg once daily for three days. Plasma was collected from these mice 4 hours after receiving the last dose of LOVENOX. In the third cohort, ISIS 404071 was administered subcutaneously to BALB/c mice at a dosage of 20 mg/kg twice a week for 3 weeks, and 2 days after receiving the last dose of ISIS 404071, LOVENOX was administered subcutaneously at a dosage of 20 mg/kg once daily. Plasma was collected from these mice 4 hours after the last dose of LOVENOX. In a fourth cohort, PBS was administered subcutaneously twice a week for three weeks, as a control. Plasma was collected from these mice 5 days after the last dose.

PT and aPTT Assay

PT and aPTT values provided in Table 31 are reported as International Normalized Ratio (INR) values. As shown in Table 31, PT is not significantly affected by treatment with ISIS 404071, LOVENOX, or treatment with ISIS 40471 combined with LOVENOX. These data suggest that there is no combinational effect on PT by ISIS 404071 combined with LOVENOX. Also shown in Table 31, treatment with LOVENOX and treatment with ISIS 404071 combined with LOVENOX increase aPTT. These data suggest that the combined treatment of ISIS 404071 and LOVENOX has an additive effect on aPTT.

TABLE 31 Effect of combination of ISIS 404071 and LOVENOX on PT and aPTT in murine plasma PT INR aPTT INR ISIS 404071 0.95 1.31 LOVENOX 1.04 2.04 404071 + LOVENOX 1.04 2.58

Example 20 In Vivo Effect of Antisense Inhibition of Murine Factor 11 in Combination with Apixaban on PT and aPTT Treatment

PT and aPTT were measured using PPP from mice treated with ISIS 404071 in combination with Apixaban. In the first cohort, ISIS 404071 was administered subcutaneously to BALB/c mice at a dosage of 25 mg/kg twice a week for 3 weeks. Plasma was collected from these mice 5 days after receiving the last dose of ISIS 404071. In the second cohort, Apixaban was administered subcutaneously to BALB/c mice at a dosage of 6 mg/kg twice daily for three days. Plasma was collected from these mice 20 minutes after receiving the last dose of Apixaban. In the third cohort, ISIS 404071 was administered subcutaneously to BALB/c mice at a dosage of 20 mg/kg twice a week for 3 weeks, and Apixaban was administered subcutaneously at a dosage of 6 mg/kg twice daily on the last three days of ISIS 404071 treatment. Plasma was collected from these mice 20 minutes after receiving the last dose of Apixaban. In a fourth cohort, PBS was administered subcutaneously twice a week for three weeks, as a control. Plasma was collected 5 days after the last dose of PBS.

PT and aPTT Assay

PT and aPTT values provided in Table 32 are reported as International Normalized Ratio (INR) values. As shown in Table 32, PT is not significantly affected by treatment with ISIS 404071. However, Apixaban and Apixaban combined with ISIS 404071 increased PT. Also shown in Table 32, Apixaban, ISIS 404071, and ISIS 404071 combined with Apixaban increase aPTT.

TABLE 32 Effect of combination of ISIS 404071 and Apixaban on PT and aPTT in murine plasma PT INR aPTT INR ISIS 404071 0.95 1.31 Apixaban 3.25 1.44 404071 + Apixaban 3.50 2.26

Example 21 In Vivo Effect of Antisense Inhibition of Murine Factor 11 in Combination with Warfarin on PT and aPTT Treatment

PT and aPTT were measured using PPP from mice treated with ISIS 404071 in combination with warfarin. Two groups of BALB/c mice were treated with either 25 mg/kg or 50 mg/kg of ISIS 404071, administered subcutaneously twice a week for 3 weeks. Plasma was collected from each group 5 days after the last dose was administered. In a third group, BALB/c mice were treated with 2 mg/kg of warfarin once daily for 5 days. Plasma was collected 6 hours after the last dose of warfarin was administered. Two additional groups of BALB/c mice were treated with either 25 mg/kg or 50 mg/kg of ISIS 404071, administered subcutaneously twice a week for 3 weeks and warfarin was administered subcutaneously at a dosage of 2 mg/kg once daily on the last 5 days of ISIS 404071 treatment. Plasma was collected from each group 6 hours after the last warfarin treatment. In a final group of BALB/c mice, PBS was administered subcutaneously twice a week for three weeks, as a control. Plasma was collected 5 days after the last PBS treatment.

PT and aPTT Assay

PT and aPTT values provided in Table 33 are reported as International Normalized Ratio (INR) values. As shown in Table 33, PT is not affected by treatment with PBS or ISIS 404071 at either dosage. However, treatment with 2 mg/kg warfarin, 25 mg/kg ISIS 404071 in combination with 2 mg/kg warfarin, and 50 mg/kg ISIS 404071 in combination with 2 mg/kg warfarin increase PT. These data suggest that the combined treatment of ISIS 404071 and warfarin has an additive effect on PT. Also shown in Table 33, aPTT is affected by treatment with ISIS 404071 and warfarin. The combination of ISIS 404071 and warfarin show an increase in aPTT greater than either drug alone. These data suggest that the combined treatment of ISIS 404071 and warfarin has a synergistic effect on aPTT.

TABLE 33 Effect of combination of ISIS 404071 and warfarin on PT and aPTT in murine plasma Dose in mg/kg PT INR aPTT INR ISIS 404071 25 0.98 1.37 50 0.93 1.49 Warfarin 2 21.33 2.52 ISIS 404071(+Warfarin) 25 25.77 4.45 50 36.33 4.75

Example 22 In Vivo Antithrombotic Effect of Antisense Inhibition of Murine FACTOR 11 on Mesenteric Vein Thrombosis in Mice Treatment

In a first cohort, ISIS 404071 was administered subcutaneously to C57BL/6 mice twice a week for three weeks at a dose of 50 mg/kg. In a second cohort, a control oligonucleotide, ISIS 405277 (AAGGACCTACACTATGGAAT; antisense oligonucleotide for Factor 2), incorporated herein as SEQ ID NO: 12 was administered subcutaneously to C57B1/6 mice twice a week for three weeks at a dose of 50 mg/kg.

Platelet Preparation

Blood was collected from the retro-orbital venous plexus of naïve C57BL/6 mice by puncture and collected in polypropylene tubes containing 300 μl of heparin (30 U/ml). Platelet rich plasma (PRP) was obtained by centrifugation at 1000 rpm for 5 min. The PRP was transferred to fresh tubes containing 2 μl of Prostaglandin I₂ (PGI₂) (2 μg/ml) and incubated at 37° C. for 5 min. After centrifugation at 2600 rpm, pellets were resuspended in 1 ml modified Tyrode's-HEPES buffer (137 mM NaCl, 0.3 mM Na₂HPO₄, 2 mM KCl, 12 mM NaHCO₃, 5 mM HEPES, 5 mM glucose, 0.35% BSA, pH 7.2) containing 2 μl of PGI₂ and incubated at 37° C. for 5 min. The suspended pellet was centrifuged at 2600 rpm for 5 min. To remove PGI₂, the washing step was repeated twice and platelets were fluorescently labeled with calcein AM 2.5 μg/mL (Molecular Probes, Eugene, Oreg.) for 10 min at room temperature.

Intravital Microscopy for Thrombosis

Fluorescently-labeled platelets were injected intravenously in ISIS 404071 treated and control oligonucleotide treated C57BL/6 mice. The mice were anaesthetized with 2.5% avertin, and an incision was made through the abdominal wall to expose mesenteric veins 250-300-μm in diameter and having a shear rate of approximately 150 s⁻¹. The exposed mesentery was kept moist throughout the experiment by periodic superfusion with warmed (3TC) PBS. The mesentery was transluminated with a 12V, 100 W, DC stabilized source. Veins were visualized using a Zeiss (Germany) Axiovert 135 inverted microscope (Objective 32×) connected to an SVHS video recorder (AG-6730; Panasonic, Tokyo, Japan) using a CCD video camera (Hamamatsu Photonic Systems, Hamamatsu City, Japan). Centerline erythrocyte velocity (V_(rbc)) was measured using an optical Doppler velocimeter (Microcirculation Research Institute, Texas A&M College of Medicine, College Station, Tex.). Venular shear rate (τ) was calculated based on Poiseuille's Law for a newtonian fluid, τ=8(V_(mean)/D_(v)), where D_(v) is the diameter of the venule and V_(mean) is estimated from the measured V_(rbc) using the empirical correlation V_(mean)=V_(rbc)/1.6.

Results Analysis

Mesenteric vein thrombosis was performed two days after the last antisense oligonucleotide injection. Thrombosis was induced by applying Whatman paper soaked in a 10% FeCl₃ solution for 5 minutes on the mesenteric vein. The vein was monitored for 40 minutes, or until occlusion. The elapsed time before the first thrombus 30-50 μm in diameter and the elapsed time before blood stopped flowing for 30 seconds were observed.

Thrombus formation (30 μm in diameter) occurred in mice treated with ISIS 404071 at 14.8±1.7 minutes. Thrombus formation (30 μm in diameter) occurred in control mice at 8.9±0.6 minutes. Occlusive thrombi formed in control mice at 19.3±0.8 min and all injured venules occluded. In contrast, the majority of the veins in ISIS 404071 treated mice did not occlude when observation was terminated 40 minutes after injury and those veins showing occlusion. The only vein showing occlusion in the ISIS 404071 treated mice occluded at 29.5 minutes and reopened after 5 minutes, prior to the end of the study.

Example 23 In Vivo Sense-Oligonucleotide-Antidote for Antisense Inhibition of Murine Factor 11 in BALB/c Mice Treatment

The effect of the specific sense oligonucleotide to ISIS 404071 as an antidote was tested in BALB/c mice. In a first cohort, ISIS 404071 was administered subcutaneously to BALB/c mice twice a week for three weeks at a dose of 40 mg/kg. In a second cohort, ISIS 404057 was administered subcutaneously to BALB/c mice twice a week for three weeks at a dose of 40 mg/kg. The ISIS 404071 specific antidote, ISIS 418026 (CCTCTGAAAGTGGATTACCA; complementary to ISIS 404071), incorporated herein as SEQ ID NO: 13, was administered to both cohorts subcutaneously in a single injection of 90 mg/kg 48 hours after the final treatment of ISIS 404071 or 404057. In a third cohort, ISIS 404071 was administered subcutaneously to BALB/c mice twice a week for three weeks at a dose of 40 mg/kg. Following the last treatment of ISIS 404071, mice were injected subcutaneously injected with PBS. In a fourth cohort, ISIS 404057 was administered subcutaneously to BALB/c mice twice a week for three weeks at a dose of 40 mg/kg. Following the last treatment of ISIS 404057, mice were injected subcutaneously injected with PBS. Following antidote administration, a set of 4 mice from each cohort were sacrificed at 12 hours, 1 day, 2 days, 3 days, 7 days, and 14 days. Whole liver was collected for RNA analysis and PPP was collected for aPTT analysis.

RNA Analysis

RNA was extracted from liver tissue for real-time PCR analysis of Factor 11. Results are presented as percent inhibition of Factor 11, relative to PBS control. As shown in Table 34, mice treated with ISIS 404071 without antidote showed progressive decrease in inhibition over the 14 day observation period. However, mice treated with ISIS 404071 and antidote showed an accelerated decrease in inhibition over the 14 day observation period in comparison to mice which did not receive antidote. Also shown in Table 34, treatment with ISIS 418026 had no effect on inhibition of Factor 11 mRNA expression in ISIS 404057 treated mice.

TABLE 34 Percent inhibition of mouse Factor 11 mRNA compared to PBS control 12 hours 1 day 2 days 3 days 7 days 14 days ISIS 404071 93 90 89 88 81 67 ISIS 404071 + 90 87 72 66 57 31 ISIS 418026 ISIS 404057 n.d. n.d. n.d. 95 n.d. n.d. ISIS 404057 + n.d. n.d. n.d. 97 n.d. n.d. ISIS 418026 n.d. = no data aPTT Assay

As shown in Table 35, mice treated with ISIS 404071 and antidote (ISIS 418026) showed progressive decrease of aPTT over the 14 day observation period compared to mice treated with ISIS 404071 without antidote.

TABLE 35 Effect of antidote treatment on aPTT INR 12 hours 1 day 2 day 3 day 7 day 14 day ISIS 404071 1.51 1.30 1.35 1.27 1.18 1.05 ISIS 404071 + 1.45 1.23 1.16 1.15 1.10 0.95 ISIS 418026

Example 24 In Vivo Factor 7a Protein-Antidote for Antisense Inhibition of Murine Factor 11 in BALB/c Mice Treatment

The effect of human Factor 7a (Factor VIIa) protein as an antidote for ISIS 404071 was tested in BALB/c mice. Two experimental groups of BALB/c mice were treated with 20 mg/kg of ISIS 404071, administered subcutaneously twice a week for 3 weeks. Two control groups of BALB/c mice were treated with PBS, administered subcutaneously twice a week for 3 weeks. Thrombus formation was induced with FeCl₃ in all of the mice except the first control group. Fifteen minutes before FeCl₃ treatment, the first experimental group was treated with 5 μg/kg of human Factor 7a protein antidote (product no. 407act, American Diagnostica Inc.). Two days after their last dose, all mice were anesthetized with 150 mg/kg of ketamine mixed with 10 mg/kg of xylazine administered by intraperitoneal injection.

In mice undergoing FeCl₃ treatment, thrombus formation was induced by applying a piece of filter paper (2×4 mm) pre-saturated with 10% FeCl₃ solution directly on the vena cava. After 3 minutes of exposure, the filter paper was removed. Thirty minutes after the filter paper application, a fixed length of the vein containing the thrombus was dissected out for platelet analysis.

Quantification of Platelet Composition

Real-time PCR quantification of platelet factor-4 (PF-4) was used to quantify platelets in the vena cava as a measure of thrombus formation. Results are presented as a percentage of PF-4 in antidote treated and untreated mice, as compared to the two PBS-treated control groups. As shown in Table 36, animals treated with human Factor 7a protein antidote expressed more PF-4 in comparison to animals treated with ISIS 404071 alone. These data indicate that human Factor 7a is successful in rescuing the effect of antisense oligonucleotide inhibition.

TABLE 36 Analysis of thrombus formation by real-time PCR quantification of PF-4 in the FeCl₃ induced venous thrombosis model Treatment PF-4 PBS − FeCl₃ 0 PBS + FeCl₃ 100 ISIS 404071 18 ISIS 404071 + hFV7a 68

Example 25 In Vivo Antisense Inhibition of Murine Factor 11 in the Collagenase-Induced Intracerebral Hemorrhage Model Treatment

ISIS 404071 and warfarin (COUMADIN) were examined in the collegenase-induced intracerebral hemorrhage model. In a first cohort, ISIS 404071 was administered subcutaneously to BALB/c mice twice a week for two weeks at a dose 40 mg/kg. In a second cohort, warfarin was administered intraperioneally to mice twice a week for two weeks at a dose of 2 mg/kg. In a third cohort, ISIS 421208 (TCGGAAGCGACTCTTATATG, 8 mismatches to murine Factor 11, incorporated herein as SEQ ID NO: 14) was administered subcutaneously to BALB/c mice twice a week for two weeks at a dose 40 mg/kg. In a fourth cohort, PBS was administered to BALB/c mice twice a week for two weeks.

Two days after receiving their final dose, all mice in all cohorts were anesthetized with 5 μg/g of avertin. Next, the mice were injected at −1 mm AP, 1 mm R ML, −4 mm DV from bregma flat skull with a 10 μL Hamilton syringe containing 0.075 U collagenase (150 U/mL). Collagenase was delivered over 5 minutes and the needle was kept in place for an additional 5 minutes to prevent reflux. The mice were then analyzed for hemorrhagic size, neurologic deficit score, and mortality.

Table 37 presents the hemorrhage volume detected in mice after collagenase treatment, Table 38 presents the neurologic deficit score of the mice, and Table 39 presents the mortality rate of the mice. Neurological deficit is measured by a standard scoring system where no deficiency is zero and severe deficit is five. Collectively, the data suggest that ISIS 404071 did not have a significant effect on the hemorrhagic size, neurologic deficit score, or mortality of the mice. Thus, risk of intracerebral hemorrhage (a risk factor for warfarin treated individuals) is significantly reduced in ISIS 404071 treated mice in comparison to warfarin treated mice.

TABLE 37 Hemorrhagic volume after collagenase treatment Volume (mm³) PBS 51 ISIS 421208 41 ISIS 404071 38

TABLE 38 Neurologic Deficit Score after collagenase treatment Score PBS 2.4 ISIS 421208 2.0 ISIS 404071 3.8

TABLE 39 Mortality after collagenase treatment % mortality PBS 0 ISIS 421208 0 ISIS 404071 20 Warfarin 80

Example 26 In Vivo Effect of Antisense Inhibition of Murine Factor 11 in Combination with PLAVIX in the FeCl₃ Induced Venous Thrombosis (VT) Model Treatment

The combination of ISIS 404071 and PLAVIX was evaluated in the FeCl₃ induced VT mouse model. Four groups of eight BALB/c mice, weighing approximately 25 g each, were treated with 6.25 mg/kg, 12.50 mg/kg, 25.00 mg/kg, or 50.00 mg/kg of PLAVIX. Mice were given two doses of PLAVIX on day one and one dose of PLAVIX on day two, two hours before surgery.

An additional four groups of eight BALB/c mice, weighing approximately 25 g each, were treated with 20 mg/kg of ISIS 404071, administered subcutaneously twice a week for three weeks. After the last dose of ISIS 404071, mice were treated with 6.25 mg/kg, 12.50 mg/kg, 25.00 mg/kg, or 50.00 mg/kg of PLAVIX. Two doses of PLAVIX were administered to the mice on day one and one dose of PLAVIX was administered on day two, two hours before surgery.

Two control groups of eight BALB/c mice, weighing approximately 25 g each, were not treated with ISIS 404071 or PLAVIX. An additional two control groups of eight BALB/c mice, weighing approximately 25 g each, were treated with 20 mg/kg of ISIS 404071, administered subcutaneously twice a week for three weeks, but were not treated with PLAVIX. Thrombus formation was induced with FeCl₃ in all of the mice except the first and third control groups. All mice were anesthetized with 150 mg/kg of ketamine mixed with 10 mg/kg of xylazine administered by intraperitoneal injection.

In mice undergoing FeCl₃ treatment, thrombus formation was induced by applying a piece of filter paper (2×4 mm) pre-saturated with 10% FeCl₃ solution directly on the inferior vena cava. After 3 minutes of exposure, the filter paper was removed. Thirty minutes after the filter paper application, a fixed length of the vein containing the thrombus was dissected out for platelet analysis.

Quantification of Platelet Composition

Real-time PCR quantification of PF-4 was used to quantify platelets in the vena cava as a measure of thrombus formation. As shown in Table 40, treatment with PLAVIX resulted in a reduction of PF-4 in comparison to the PBS control. Treatment with PLAVIX in combination with ISIS 404071 resulted in a higher reduction of PF-4 in comparison to PLAVIX alone. Therefore, the combination of anti-platelet therapy with Factor 11 ASO increases antithrombotic activity. Data is presented as percent of PF-4 mRNA as compared to the PBS+FeCl₃ control.

TABLE 40 Analysis of thrombus formation by real-time PCR quantification of PF-4 in the FeCl3 induced venous thrombosis model ISIS 404071 PLAVIX Treatment mg/kg mg/kg PF-4 PBS − FeCl₃ 0 0 29 PBS + FeCl₃ 0 0 100 PLAVIX only 0 6.25 59 0 12.50 37 0 25.00 30 0 50.00 30 ISIS 404071 − FeCl₃ 20 0 27 ISIS 404071 + FeCl₃ 20 0 40 PLAVIX (+ISIS 20 6.25 35 404071) 20 12.50 38 20 25.00 25 20 50.00 35

Example 27 In Vivo Effect of Antisense Inhibition of Murine Factor 11 in Combination with PLAVIX on Bleeding Treatment

Tail-bleeding was measured to observe whether treatment with ISIS 404071 in combination with PLAVIX causes an increase in bleeding tendency. ISIS 404071 was administered subcutaneously at a dosage of 20 mg/kg twice a week for 3 weeks to 5 groups of eight BALB/c mice. After the last dose of ISIS 404071, mice were treated with 0 mg/kg, 6.25 mg/kg, 12.50 mg/kg, 25.00 mg/kg, or 50.00 mg/kg of PLAVIX. Two doses of PLAVIX were administered to the mice on day one and one dose of PLAVIX was administered on day two, two hours before bleeding.

An additional 5 groups of eight BABL/c mice were treated similarly, except they did not receive ISIS 404071 injections.

Tail-Bleeding Assay

Two hours after receiving their final treatment, mice were placed in a tail bleeding chamber. Mice were anesthetized in the chamber with isoflurane and a small piece of tail (approximately 4 mm from the tip) was cut with sterile scissors. The cut tail was immediately placed in a 15 mL Falcon tube filled with approximately 10 mL of 0.9% NaCl buffer solution warmed to 37° C. The blood was collected for the course of 40 minutes. The saline filled tubes were weighed both before and after bleeding.

Taken with the results of Example 26, these data show that the combination of anti-platelet therapy with Factor 11 ASO increases antithrombotic activity without increased bleeding risk.

TABLE 41 Tail bleeding assay comparing PLAVIX and the combination of PLAVIX and ISIS 404071 ISIS 404071 PLAVIX Blood Treatment mg/kg mg/kg (g) No treatment 0 0 0.040 PLAVIX only 0  6.25 mg/kg 0.075 0 12.50 mg/kg 0.205 0 25.00 mg/kg 0.524 0 50.00 mg/kg 0.628 ISIS 404071 only 20 mg/kg 0 0 PLAVIX (+ISIS 20 mg/kg  6.25 mg/kg 0.065 404071) 20 mg/kg 12.50 mg/kg 0.300 20 mg/kg 25.00 mg/kg 0.401 20 mg/kg 50.00 mg/kg 0.577

Example 28 In Vivo Effect of a Factor Xa Small Molecule Inhibitor in Combination with PLAVIX on Bleeding Treatment

Tail-bleeding was measured to observe whether treatment with a Factor 10a small molecule in combination with PLAVIX causes an increase in bleeding tendency. Five groups of eight BALB/c mice were treated with 0 mg/kg, 6.25 mg/kg, 12.50 mg/kg, 25.00 mg/kg, or 50.00 mg/kg of PLAVIX. Mice were given two doses of PLAVIX on day one and one dose of PLAVIX on day two, two hours before bleeding.

An additional five groups of eight BALB/c mice were treated with 0 mg/kg, 6.25 mg/kg, 12.50 mg/kg, 25.00 mg/kg, or 50.00 mg/kg of PLAVIX. Mice were given two doses of PLAVIX on day one and one dose of PLAVIX on day two, two hours before bleeding. These mice were also treated with 0.5 mg/kg of Apixaban, a small molecule Factor 10a inhibitor, intraperitoneally one time 20 minutes before bleeding.

Tail-Bleeding Assay

Two hours after receiving their final treatment, mice were placed in a tail bleeding chamber. Mice were anesthetized in the chamber with isoflurane and a small piece of tail (approximately 4 mm from the tip) was cut with sterile scissors. The cut tail was immediately placed in a 15 mL Falcon tube filled with approximately 10 mL of 0.9% NaCl buffer solution warmed to 37° C. The blood was collected for the course of 40 minutes. The saline filled tubes were weighed both before and after bleeding.

As shown below in Table 42, these data show that the combination of anti-platelet therapy with a small molecule Factor 10a inhibitor, such as Apixaban, increases bleeding risk. Therefore, treatment with the combination of anti-platelet therapy with a Factor 11 ASO provides a better safety profile in comparison to the safety profile of a combination of anti-platelet therapy with a small molecule Factor 10a inhibitor.

TABLE 42 Tail bleeding assay comparing PLAVIX, Apixaban, and the combination of PLAVIX and Apixaban Apixaban PLAVIX Blood Treatment mg/kg mg/kg (g) No treatment 0 0 0.002 PLAVIX only 0  6.25 mg/kg 0.061 0 12.50 mg/kg 0.149 0 25.00 mg/kg 0.246 0 50.00 mg/kg 0.258 Apixaban only 0.5 mg/kg 0 0.004 PLAVIX (+Apixaban) 0.5 mg/kg  6.25 mg/kg 0.258 0.5 mg/kg 12.50 mg/kg 0.252 0.5 mg/kg 25.00 mg/kg 0.361 0.5 mg/kg 50.00 mg/kg 0.363

Example 29 Time Course of In Vivo, Antisense-Mediated Reduction of Murine Factor 11 and Corresponding Anticoagulation in Blood Treatment

The time course of antisense-mediated reduction of murine Factor 11 mRNA was observed in BALB/c mice. One dose of 50 mg/kg ISIS 404071 was administered subcutaneously to BALB/c mice. Following ISIS 404071 administration, mice were sacrificed at 12 hours, 1 day, 2 days, 3 days, 4 days, 7 days, 14 days, 28 days, and 56 days. Whole liver was collected for RNA analysis and PPP was collected for aPTT analysis. A control group of mice was treated with one subcutaneous dose of PBS.

RNA Analysis

RNA was extracted from liver tissue for real-time PCR analysis of Factor 11. Results are presented relative to PBS control. Mice treated with ISIS 404071 showed significant Factor 11 mRNA down-regulation by day 1. Mice began regaining Factor 11 mRNA expression by day 14. Mice regained full Factor 11 mRNA expression by day 28 and results from day 56 indicate that Factor mRNA was maintained at pre-treatment levels. Therefore, ISIS 404071 treated mice did not experience a rebound effect.

The rebound effect has been previously observed in antibody-mediated reduction of Factor 11 (Blood, First Edition Paper, prepublished online Oct. 22, 2008; Prevention of vascular graft occlusion and thrombus-associated thrombin generation by inhibition of factor XI). Because over expression of Factor 11 can be damaging by leading to increased coagulation, these data suggest that antisense-mediated inhibition of Factor 11 is safer than antibody-mediated inhibition of Factor 11 since antisense-mediated inhibition of Factor 11 does not rebound.

aPTT Assay

aPTT values provided in Table 43 are reported as International Normalized Ratio (INR) values. INR values for aPTT were determined by dividing the aPTT value for ISIS 404071 treated mice by the aPTT for the PBS treated group. This ratio was then raised to the power of the International Sensitivity Index (ISI) of the tissue factor used. As shown in Table 43, mice treated with ISIS 404071 showed progressive decrease of aPTT until day 4 and then progressive increase to pre-treatment levels from day 7 to day 28.

TABLE 43 Effect of ISIS 404071 treatment on aPTT INR* 12 hours day 1 day 2 day 3 day 4 day 7 day 14 day 28 day 56 ISIS 404071 0 1.02 1.12 1.29 1.30 1.25 1.11 1.02 0 *values in Table 43 are approximate

Example 30 Antisense Inhibition of Human Factor 11 in HepG2 Cells by Oligonucleotides Designed by Microwalk

Additional gapmers were designed based on ISIS 416850 and ISIS 416858 (see Table 8 above). These gapmers were shifted slightly upstream and downstream (i.e. “microwalk”) of ISIS 416850 and ISIS 416858. The microwalk gapmers were designed with either 5-8-5 MOE or 6-8-6 MOE motifs.

These microwalk gapmers were tested in vitro. Cultured HepG2 cells at a density of 20,000 cells per well were transfected using electroporation with 8,000 nM antisense oligonucleotide. After a treatment period of approximately 24 hours, RNA was isolated from the cells and Factor 11 mRNA levels were measured by quantitative real-time PCR. Factor 11 mRNA levels were adjusted according to total RNA content, as measured by RIBOGREEN. Results are presented as percent inhibition of Factor 11, relative to untreated control cells.

ISIS 416850 and ISIS 416858, as well as selected gapmers from Tables 1 and 8 (i.e., ISIS 412206, ISIS 412223, ISIS 412224, ISIS 412225, ISIS 413481, ISIS 413482, ISIS 416825, ISIS 416848, ISIS 416849, ISIS 416850, ISIS 416851, ISIS 416852, ISIS 416853, ISIS 416854, ISIS 416855, ISIS 416856, ISIS 416857, ISIS 416858, ISIS 416859, ISIS 416860, ISIS 416861, ISIS 416862, ISIS 416863, ISIS 416864, ISIS 416865, ISIS 416866, and ISIS 416867) were retested in vitro along with the microwalk gapmers under the same condition as described above.

The chimeric antisense oligonucleotides in Table 44 were designed as 5-10-5 MOE, 5-8-5 and 6-8-6 MOE gapmers. The first two listed gapmers in Table 44 are the original gapmers (ISIS 416850 and ISIS 416858) from which ISIS 445493-445543 were designed via microwalk, and are designated by an asterisk. The 5-10-5 gapmers are 20 nucleotides in length, wherein the central gap segment is comprised of ten 2′-deoxynucleotides and is flanked on both sides (in the 5′ and 3′ directions) by wings comprising five nucleotides each. The 5-8-5 gapmers are 18 nucleotides in length, wherein the central gap segment is comprised of eight 2′-deoxynucleotides and is flanked on both sides (in the 5′ and 3′ directions) by wings comprising five nucleotides each. The 6-8-6 gapmers are 20 nucleotides in length, wherein the central gap segment is comprised of eight 2′-deoxynucleotides and is flanked on both sides (in the 5′ and 3′ directions) by wings comprising six nucleotides each. For each of the motifs (5-10-5, 5-8-5 and 6-8-6), each nucleotide in the 5′ wing segment and each nucleotide in the 3′ wing segment has a 2′-MOE modification. The internucleoside linkages throughout each gapmer are phosphorothioate (P═S) linkages. All cytidine residues throughout each gapmer are 5-methylcytidines. “Human Target start site” indicates the 5′-most nucleotide to which the gapmer is targeted in the human sequence. “Human Target stop site” indicates the 3′-most nucleotide to which the gapmer is targeted in the human sequence. Each gapmer listed in Table 44 is targeted to SEQ ID NO: 1 (GENBANK Accession No. NM_(—)000128.3). Each gapmer is Table 44 is also fully cross-reactive with the rhesus monkey Factor 11 gene sequence, designated herein as SEQ ID NO: 274 (exons 1-15 GENBANK Accession No. NW_(—)001118167.1). ‘Rhesus monkey start site’ indicates the 5′-most nucleotide to which the gapmer is targeted in the rhesus monkey sequence. ‘Rhesus monkey stop site’ indicates the 3′-most nucleotide to which the gapmer is targeted to the rhesus monkey sequence.

As shown in Table 44, all of the microwalk designed gapmers targeted to the target region beginning at the target start site 1275 and ending at the target stop site 1317 (i.e. nucleobases 1275-1317) of SEQ ID NO: 1 exhibited at least 60% inhibition of Factor 11 mRNA. Similarly, all of the re-tested gapmers from Tables 1 and 8 exhibited at least 60% inhibition.

Several of the gapmers exhibited at least 70% inhibition, including ISIS numbers: ISIS 412206, 412224, 412225, 413481, 413482, 416825, 416848, 416849, 416850, 416851, 416852, 416853, 416854, 416855, 416856, 416857, 416858, 416859, 416860, 416861, 416862, 416863, 416864, 416865, 416866, 416867, 445494, 445495, 445496, 445497, 445498, 445499, 445500, 445501, 445502, 445503, 445504, 445505, 445506, 445507, 445508, 445509, 445510, 445511, 445512, 445513, 445514, 445515, 445516, 445517, 445518, 445519, 445520, 445521, 445522, 445523, 445524, 445525, 445526, 445527, 445528, 445529, 445530, 445531, 445532, 445533, 445534, 445535, 445536, 445537, 455538, 445539, 445540, 445541, 445542, and 445543.

Several of the gapmers exhibited at least 80% inhibition, including ISIS numbers: ISIS 412206, 412224, 412225, 413481, 413482, 416825, 416848, 416849, 416850, 416851, 416852, 416853, 416854, 416855, 416856, 416857, 416858, 416859, 416860, 416861, 416862, 416863, 416864, 416865, 416866, 416867, 445494, 445495, 445496, 445497, 445498, 445500, 445501, 445502, 445503, 445504, 445505, 445506, 445507, 445508, 445509, 445510, 445513, 445514, 445519, 445520, 445521, 445522, 445525, 445526, 445529, 445530, 445531, 445532, 445533, 445534, 445535, 445536, 455538, 445541, and 445542.

Several of the gapmers exhibited at least 90% inhibition, including ISIS numbers: ISIS 412206, 416825, 416850, 416857, 416858, 416861, 445522, and 445531.

TABLE 44 Inhibition of human Factor 11 mRNA levels by chimeric antisense oligonucleotides targeted to SEQ ID NO: 1 (GENBANK Accession No. NM_000128.3) Rhesus Rhesus Human Human SEQ monkey monkey Start Stop Sequence Percent ID Start Stop ISIS No. Site Site (5′ to 3′) inhibition Motif No. Site Site *416850 1278 1297 TGCACAGTTT 91 May 10 2005 215 1277 1296 CTGGCAGGCC *416858 1288 1307 ACGGCATTGG 90 May 10 2005 223 1287 1306 TGCACAGTTT 416825 680 699 GCCCTTCATGT 90 May 10 2005 190 679 698 CTAGGTCCA 412206 738 757 CCGTGCATCTT 91 May 10 2005 34 737 756 TCTTGGCAT 412223 1275 1294 ACAGTTTCTG 62 May 10 2005 51 1274 1293 GCAGGCCTCG 445493 1275 1294 ACAGTTTCTG 69 Jun. 8 2006 51 1274 1293 GCAGGCCTCG 445518 1275 1292 AGTTTCTGGC 75 May 8 2005 242 1274 1291 AGGCCTCG 416848 1276 1295 CACAGTTTCT 87 May 10 2005 213 1275 1294 GGCAGGCCTC 445494 1276 1295 CACAGTTTCT 85 Jun. 8 2006 213 1275 1294 GGCAGGCCTC 445519 1276 1293 CAGTTTCTGG 81 May 8 2005 243 1275 1292 CAGGCCTC 416849 1277 1296 GCACAGTTTC 88 May 10 2005 214 1276 1295 TGGCAGGCCT 445495 1277 1296 GCACAGTTTC 89 Jun. 8 2006 214 1276 1295 TGGCAGGCCT 445520 1277 1294 ACAGTTTCTG 82 May 8 2005 244 1276 1293 GCAGGCCT 445496 1278 1297 TGCACAGTTT 87 Jun. 8 2006 215 1277 1296 CTGGCAGGCC 445521 1278 1295 CACAGTTTCT 87 May 8 2005 245 1277 1294 GGCAGGCC 416851 1279 1298 GTGCACAGTT 89 May 10 2005 216 1278 1297 TCTGGCAGGC 445497 1279 1298 GTGCACAGTT 81 Jun. 8 2006 216 1278 1297 TCTGGCAGGC 445522 1279 1296 GCACAGTTTC 91 May 8 2005 246 1278 1295 TGGCAGGC 413481 1280 1299 GGTGCACAGT 82 May 10 2005 114 1279 1298 TTCTGGCAGG 445498 1280 1299 GGTGCACAGT 83 Jun. 8 2006 114 1279 1298 TTCTGGCAGG 445523 1280 1297 TGCACAGTTT 73 May 8 2005 267 1279 1296 CTGGCAGG 416852 1281 1300 TGGTGCACAG 87 May 10 2005 217 1280 1299 TTTCTGGCAG 445499 1281 1300 TGGTGCACAG 75 Jun. 8 2006 217 1280 1299 TTTCTGGCAG 445524 1281 1298 GTGCACAGTT 75 May 8 2005 247 1280 1297 TCTGGCAG 416853 1282 1301 TTGGTGCACA 84 May 10 2005 218 1281 1300 GTTTCTGGCA 445500 1282 1301 TTGGTGCACA 81 Jun. 8 2006 218 1281 1300 GTTTCTGGCA 445525 1282 1299 GGTGCACAGT 85 May 8 2005 248 1281 1298 TTCTGGCA 416854 1283 1302 ATTGGTGCAC 86 May 10 2005 219 1282 1301 AGTTTCTGGC 445501 1283 1302 ATTGGTGCAC 83 Jun. 8 2006 219 1282 1301 AGTTTCTGGC 445526 1283 1300 TGGTGCACAG 81 May 8 2005 249 1282 1299 TTTCTGGC 416855 1284 1303 CATTGGTGCA 85 May 10 2005 220 1283 1302 CAGTTTCTGG 445502 1284 1303 CATTGGTGCA 83 Jun. 8 2006 220 1283 1302 CAGTTTCTGG 445527 1284 1301 TTGGTGCACA 70 May 8 2005 250 1283 1300 GTTTCTGG 412224 1285 1304 GCATTGGTGC 84 May 10 2005 52 1284 1303 ACAGTTTCTG 445503 1285 1304 GCATTGGTGC 89 Jun. 8 2006 52 1284 1303 ACAGTTTCTG 445528 1285 1302 ATTGGTGCAC 73 May 8 2005 251 1284 1301 AGTTTCTG 416856 1286 1305 GGCATTGGTG 84 May 10 2005 221 1285 1304 CACAGTTTCT 445504 1286 1305 GGCATTGGTG 87 Jun. 8 2006 221 1285 1304 CACAGTTTCT 445529 1286 1303 CATTGGTGCA 85 May 8 2005 252 1285 1302 CAGTTTCT 416857 1287 1306 CGGCATTGGT 91 May 10 2005 222 1286 1305 GCACAGTTTC 445505 1287 1306 CGGCATTGGT 89 Jun. 8 2006 222 1286 1305 GCACAGTTTC 445530 1287 1304 GCATTGGTGC 83 May 8 2005 253 1286 1303 ACAGTTTC 445506 1288 1307 ACGGCATTGG 86 Jun. 8 2006 223 1287 1306 TGCACAGTTT 445531 1288 1305 GGCATTGGTG 90 May 8 2005 254 1287 1304 CACAGTTT 416859 1289 1308 GACGGCATTG 85 May 10 2005 224 1288 1307 GTGCACAGTT 445507 1289 1308 GACGGCATTG 85 Jun. 8 2006 224 1288 1307 GTGCACAGTT 445532 1289 1306 CGGCATTGGT 89 May 8 2005 255 1288 1305 GCACAGTT 413482 1290 1309 GGACGGCATT 88 May 10 2005 115 1289 1308 GGTGCACAGT 445508 1290 1309 GGACGGCATT 81 Jun. 8 2006 115 1289 1308 GGTGCACAGT 445533 1290 1307 ACGGCATTGG 87 May 8 2005 256 1289 1306 TGCACAGT 416860 1291 1310 CGGACGGCAT 89 May 10 2005 225 1290 1309 TGGTGCACAG 445509 1291 1310 CGGACGGCAT 84 Jun. 8 2006 225 1290 1309 TGGTGCACAG 445534 1291 1308 GACGGCATTG 82 May 8 2005 257 1290 1307 GTGCACAG 416861 1292 1311 GCGGACGGCA 90 May 10 2005 226 1291 1310 TTGGTGCACA 445510 1292 1311 GCGGACGGCA 88 Jun. 8 2006 226 1291 1310 TTGGTGCACA 445535 1292 1309 GGACGGCATT 83 May 8 2005 258 1291 1308 GGTGCACA 416862 1293 1312 AGCGGACGGC 89 May 10 2005 227 1292 1311 ATTGGTGCAC 445511 1293 1312 AGCGGACGGC 77 Jun. 8 2006 227 1292 1311 ATTGGTGCAC 445536 1293 1310 CGGACGGCAT 82 May 8 2005 259 1292 1309 TGGTGCAC 416863 1294 1313 CAGCGGACGG 86 May 10 2005 228 1293 1312 CATTGGTGCA 445512 1294 1313 CAGCGGACGG 79 Jun. 8 2006 228 1293 1312 CATTGGTGCA 445537 1294 1311 GCGGACGGCA 78 May 8 2005 260 1293 1310 TTGGTGCA 412225 1295 1314 GCAGCGGACG 86 May 10 2005 53 1294 1313 GCATTGGTGC 445513 1295 1314 GCAGCGGACG 85 Jun. 8 2006 53 1294 1313 GCATTGGTGC 445538 1295 1312 AGCGGACGGC 80 May 8 2005 261 1294 1311 ATTGGTGC 416864 1296 1315 GGCAGCGGAC 88 May 10 2005 229 1295 1314 GGCATTGGTG 445514 1296 1315 GGCAGCGGAC 81 Jun. 8 2006 229 1295 1314 GGCATTGGTG 445539 1296 1313 CAGCGGACGG 79 May 8 2005 262 1295 1312 CATTGGTG 416865 1297 1316 TGGCAGCGGA 86 May 10 2005 230 1296 1315 CGGCATTGGT 445515 1297 1316 TGGCAGCGGA 75 Jun. 8 2006 230 1296 1315 CGGCATTGGT 445540 1297 1314 GCAGCGGACG 74 May 8 2005 263 1296 1313 GCATTGGT 416866 1298 1317 CTGGCAGCGG 84 May 10 2005 231 1297 1316 ACGGCATTGG 445516 1298 1317 CTGGCAGCGG 79 Jun. 8 2006 231 1297 1316 ACGGCATTGG 445541 1298 1315 GGCAGCGGAC 80 May 8 2005 264 1297 1314 GGCATTGG 416867 1299 1318 ACTGGCAGCG 85 May 10 2005 232 1298 1317 GACGGCATTG 445517 1299 1318 ACTGGCAGCG 74 Jun. 8 2006 232 1298 1317 GACGGCATTG 445542 1299 1316 TGGCAGCGGA 83 May 8 2005 265 1298 1315 CGGCATTG 445543 1300 1317 CTGGCAGCGG 74 May 8 2005 266 1299 1316 ACGGCATT

Example 31 Dose-Dependent Antisense Inhibition of Human Factor 11 in HepG2 Cells

Gapmers from Example 30 exhibiting in vitro inhibition of human Factor 11 were tested at various doses in HepG2 cells. Cells were plated at a density of 20,000 cells per well and transfected using electroporation with 123.46 nM, 370.37 nM, 1,111.11 nM, 3,333.33 nM and 10,000 nM concentrations of antisense oligonucleotide, as specified in Table 45. After a treatment period of approximately 16 hours, RNA was isolated from the cells and Factor 11 mRNA levels were measured by quantitative real-time PCR. Human Factor 11 primer probe set RTS 2966 was used to measure mRNA levels. Factor 11 mRNA levels were adjusted according to total RNA content, as measured by RIBOGREEN. Results are presented as percent inhibition of Factor 11, relative to untreated control cells. As illustrated in Table 45, Factor 11 mRNA levels were reduced in a dose-dependent manner in antisense oligonucleotide treated cells.

The half maximal inhibitory concentration (IC₅₀) of each oligonucleotide was calculated by plotting the concentrations of antisense oligonucleotides used versus the percent inhibition of Factor 11 mRNA expression achieved at each concentration, and noting the concentration of antisense oligonucleotide at which 50% inhibition of Factor 11 mRNA expression was achieved compared to the PBS control. IC₅₀ values are presented in Table 45.

TABLE 45 Dose-dependent antisense inhibition of human Factor 11 in HepG2 cells via transfection of oligonucleotides using electroporation 123.47 370.37 1,111.11 3,333.33 10,000.0 IC₅₀ ISIS No. nM nM nM nM nM (μM) 416849 5 5 26 57 68 2.7 416850 0 12 36 74 73 2.8 416851 13 35 36 64 72 1.5 416856 12 23 35 59 83 1.6 416857 2 20 35 62 72 2.3 416858 0 27 36 64 70 2.2 416860 0 28 39 41 40 n.d. 416861 0 15 27 66 80 2.0 445498 3 1 27 50 58 4.8 445503 0 0 22 36 60 5.9 445504 8 20 38 53 68 2.7 445505 12 30 39 59 77 1.8 445522 0 0 44 63 74 2.9 445531 8 16 52 61 77 1.8 445532 5 12 39 60 70 2.0 n.d. = no data

Example 32 Dose-Dependent Antisense Inhibition of Human Factor 11 in HepG2 Cells by Oligonucleotides Designed by Microwalk

Additional gapmers were designed based on ISIS 416850 and ISIS 416858 (see Table 8 above). These gapmers are shifted slightly upstream and downstream (i.e. microwalk) of ISIS 416850 and ISIS 416858. Gapmers designed by microwalk have 3-8-3 MOE, 4-8-4 MOE, 2-10-2 MOE, 3-10-3 MOE, or 4-10-4 MOE motifs.

These gapmers were tested at various doses in HepG2 cells. Cells were plated at a density of 20,000 cells per well and transfected using electroporation with 375 nM, 750 nM, 1,500 nM, 3,000 nM, 6,000 nM and 12,000 nM concentrations of antisense oligonucleotide, as specified in Table 47. After a treatment period of approximately 16 hours, RNA was isolated from the cells and Factor 11 mRNA levels were measured by quantitative real-time PCR. Human Factor 11 primer probe set RTS 2966 was used to measure mRNA levels. Factor 11 mRNA levels were adjusted according to total RNA content, as measured by RIBOGREEN. Results are presented as percent inhibition of Factor 11, relative to untreated control cells.

ISIS 416850, ISIS 416858, ISIS 445522, and ISIS 445531 (see Table 45 above) were re-tested in vitro along with the microwalk gapmers under the same conditions described above.

The chimeric antisense oligonucleotides in Table 46 were designed as 3-8-3 MOE, 4-8-4 MOE, 2-10-2 MOE, 3-10-3 MOE, or 4-10-4 MOE gapmers. The 3-8-3 gapmer is 14 nucleotides in length, wherein the central gap segment is comprised of eight 2′-deoxynucleotides and is flanked on both sides (in the 5′ and 3′ directions) by wings comprising three nucleotides each. The 4-8-4 gapmer is 16 nucleotides in length, wherein the central gap segment is comprised of eight 2′-deoxynucleotides and is flanked on both sides (in the 5′ and 3′ directions) by wings comprising four nucleotides each. The 2-10-2 gapmer is 14 nucleotides in length, wherein the central gap segment is comprised of ten 2′-deoxynucleotides and is flanked on both sides (in the 5′ and 3′ directions) by wings comprising two nucleotides each. The 3-10-3 gapmer is 16 nucleotides in length, wherein the central gap segment is comprised of ten 2′-deoxynucleotides and is flanked on both sides (in the 5′ and 3′ directions) by wings comprising three nucleotides each. The 4-10-4 gapmer is 18 nucleotides in length, wherein the central gap segment is comprised of ten 2′-deoxynucleotides and is flanked on both sides (in the 5′ and 3′ directions) by wings comprising four nucleotides each. For each of the motifs (3-8-3, 4-8-4, 2-10-2, 3-10-3, and 4-10-4), each nucleotide in the 5′ wing segment and each nucleotide in the 3′ wing segment has a 2′-MOE modification. The internucleoside linkages throughout each gapmer are phosphorothioate (P═S) linkages. All cytidine residues throughout each gapmer are 5-methylcytidines. “Human Target start site” indicates the 5′-most nucleotide to which the gapmer is targeted in the human sequence. “Human Target stop site” indicates the 3′-most nucleotide to which the gapmer is targeted in the human sequence. Each gapmer listed in Table 46 is targeted to SEQ ID NO: 1 (GENBANK Accession No. NM_(—)000128.3). Each gapmer is Table 46 is also fully cross-reactive with the rhesus monkey Factor 11 gene sequence, designated herein as SEQ ID NO: 274 (exons 1-15 GENBANK Accession No. NW_(—)001118167.1). ‘Rhesus monkey start site’ indicates the 5′-most nucleotide to which the gapmer is targeted in the rhesus monkey sequence. ‘Rhesus monkey stop site’ indicates the 3′-most nucleotide to which the gapmer is targeted to the rhesus monkey sequence.

TABLE 46 Chimeric antisense oligonucleotides targeted to SEQ ID NO: 1 (GENBANK Accession No. NM_000128.3) and designed by microwalk of ISIS 416850 and ISIS 416858 Human Human Rhesus Rhesus Target Target SEQ monkey monkey ISIS Start Stop ID Start Stop No. Site Site Sequence (5′ to 3′) Motif No. Site Site 449707 1280 1295 CACAGTTT 4-8-4 268 1279 1294 CTGGCAGG 449708 1281 1294 ACAGTTT 3-8-3 269 1280 1293 CTGGCAG 449709 1279 1296 GCACAGTT 4-10-4 246 1278 1295 TCTGGCAGGC 449710 1280 1295 CACAGTTT 3-10-3 268 1279 1294 CTGGCAGG 449711 1281 1294 ACAGTTT 2-10-2 269 1280 1293 CTGGCAG

Dose-response inhibition data is given in Table 47. As illustrated in Table 47, Factor 11 mRNA levels were reduced in a dose-dependent manner in antisense oligonucleotide treated cells. The IC₅₀ of each antisense oligonucleotide was also calculated and presented in Table 47. The first two listed gapmers in Table 47 are the original gapmers (ISIS 416850 and ISIS 416858) from which the remaining gapmers were designed via microwalk and are designated by an asterisk.

TABLE 47 Dose-dependent antisense inhibition of human Factor 11 in HepG2 cells via transfection of oligonucleotides using electroporation 375 750 1,500 3,000 6,000 12,000 IC₅₀ ISIS No. nM nM nM nM nM nM (μM) *416850  40 59 69 87 90 95 0.56 *416858  31 35 78 85 90 93 0.83 445522 59 71 83 82 81 92 n.d. 445531 44 64 78 86 91 93 0.44 449707 7 35 63 73 85 91 1.26 449708 0 0 22 33 61 85 4.46 449709 52 71 80 87 92 95 0.38 449710 2 21 52 70 82 87 1.59 449711 6 14 1 7 32 52 11.04 n.d. = no data

Example 33 Tolerability of Antisense Oligonucleotides Targeting Human Factor 11 in CD1 Mice

CD1 mice were treated with ISIS antisense oligonucleotides targeting human Factor 11 and evaluated for changes in the levels of various metabolic markers.

Treatment

Groups of five CD1 mice each were injected subcutaneously twice a week for 2, 4, or 6 weeks with 50 mg/kg of ISIS 416825, ISIS 416826, ISIS 416838, ISIS 416850, ISIS 416858, ISIS 416864, ISIS 416892, ISIS 416925, ISIS 416999, ISIS 417002, or ISIS 417003. A control group of five mice was injected subcutaneously with PBS for 2 weeks. All experimental groups (i.e. ASO treated mice at 2, 4, 6 weeks) were compared to the control group (i.e. PBS, 2 weeks).

Three days after the last dose was administered to all groups, the mice were sacrificed. Organ weights were measured and blood was collected for further analysis.

Organ Weight

Liver, spleen, and kidney weights were measured at the end of the study, and are presented in Tables 48, 49, and 50 as a percent of the PBS control, normalized to body weight. Those antisense oligonucleotides which did not affect more than six-fold increases in liver and spleen weight above the PBS controls were selected for further studies.

TABLE 48 Percent change in liver weight of CD1 mice after antisense oligonucleotide treatment ISIS No. 2 weeks 4 weeks 6 weeks 416825 +5 +22 +13 416826 +10 +32 +33 416838 +8 −6 0 416850 +5 +3 +6 416858 +7 +1 +10 416864 −2 +2 −5 416925 +14 +14 +33 416999 +13 +30 +47 417002 +14 +8 +35 416892 +35 +88 +95 417003 +8 +42 +32

TABLE 49 Percent change in spleen weight of CD1 mice after antisense oligonucleotide treatment ISIS No. 2 weeks 4 weeks 6 weeks 416825 −12 +19 +21 416826 −12 −5 +22 416838 +21 −8 +9 416850 −4 +6 +48 416858 −2 +8 +28 416864 −10 −2 −6 416925 −7 +33 +78 416999 +7 +22 +38 417002 +29 +26 +108 416892 +24 +30 +65 417003 +12 +101 +98

TABLE 50 Percent change in kidney weight of CD1 mice after antisense oligonucleotide treatment ISIS No. 2 weeks 4 weeks 6 weeks 416825 −12 −12 −11 416826 −13 −7 −22 416838 −2 −12 −8 416850 −10 −12 −11 416858 +1 −18 −10 416864 −4 −9 −15 416925 −4 −14 −2 416999 −9 −6 −7 417002 +3 −5 −2 416892 +2 −3 +19 417003 −9 −2 −1

Liver Function

To evaluate the effect of ISIS oligonucleotides on hepatic function, plasma concentrations of transaminases were measured using an automated clinical chemistry analyzer (Hitachi Olympus AU400e, Melville, N.Y.). Measurements of alanine transaminase (ALT) and aspartate transaminase (AST) are expressed in IU/L and the results are presented in Tables 51 and 52. Plasma levels of bilirubin and albumin were also measured using the same clinical chemistry analyzer and expressed in mg/dL. The results are presented in Tables 53 and 54. Those antisense oligonucleotides which did not affect an increase in ALT/AST levels above seven-fold of control levels were selected for further studies. Those antisense oligonucleotides which did not increase levels of bilirubin more than two-fold of the control levels were selected for further studies.

TABLE 51 Effect of antisense oligonucleotide treatment on ALT (IU/L) in CD1 mice 2 weeks 4 weeks 6 weeks PBS 36 n.d. n.d. ISIS 416825 64 314 507 ISIS 416826 182 126 1954 ISIS 416838 61 41 141 ISIS 416850 67 58 102 ISIS 416858 190 57 216 ISIS 416864 44 33 92 ISIS 416925 160 284 1284 ISIS 416999 61 160 1302 ISIS 417002 71 138 2579 ISIS 416892 66 1526 1939 ISIS 417003 192 362 2214 n.d. = no data

TABLE 52 Effect of antisense oligonucleotide treatment on AST (IU/L) in CD1 mice 2 weeks 4 weeks 6 weeks PBS 68 n.d. n.d. ISIS 416825 82 239 301 ISIS 416826 274 156 1411 ISIS 416838 106 73 107 ISIS 416850 72 88 97 ISIS 416858 236 108 178 ISIS 416864 58 46 101 ISIS 416925 144 206 712 ISIS 416999 113 130 671 ISIS 417002 96 87 1166 ISIS 416892 121 1347 1443 ISIS 417003 152 249 839 n.d. = no data

TABLE 53 Effect of antisense oligonucleotide treatment on bilirubin (mg/dL) in CD1 mice 2 weeks 4 weeks 6 weeks PBS 0.28 n.d. n.d. ISIS 416825 0.41 0.69 0.29 ISIS 416826 0.39 0.20 0.37 ISIS 416838 0.57 0.24 0.20 ISIS 416850 0.46 0.23 0.22 ISIS 416858 0.57 0.24 0.16 ISIS 416864 0.40 0.26 0.22 ISIS 416925 0.45 0.25 0.25 ISIS 416999 0.48 0.18 0.28 ISIS 417002 0.50 0.25 0.29 ISIS 416892 0.38 2.99 0.50 ISIS 417003 0.33 0.15 0.24 n.d. = no data

TABLE 54 Effect of antisense oligonucleotide treatment on albumin (mg/dL) in CD1 mice 2 weeks 4 weeks 6 weeks PBS 3.7 n.d. n.d. ISIS 416825 3.6 3.4 3.5 ISIS 416826 3.3 3.4 3.4 ISIS 416838 3.5 3.8 3.6 ISIS 416850 3.6 3.5 3.1 ISIS 416858 3.4 3.5 2.8 ISIS 416864 3.5 3.6 3.5 ISIS 416925 3.5 3.5 3.2 ISIS 416999 3.4 3.3 3.2 ISIS 417002 3.2 3.4 3.4 ISIS 416892 3.2 4.0 4.4 ISIS 417003 3.4 3.4 3.2 n.d. = no data

Kidney Function

To evaluate the effect of ISIS oligonucleotides on kidney function, plasma concentrations of blood urea nitrogen (BUN) and creatinine were measured using an automated clinical chemistry analyzer (Hitachi Olympus AU400e, Melville, N.Y.). Results are presented in Tables 55 and 56, expressed in mg/dL. Those antisense oligonucleotides which did not affect more than a two-fold increase in BUN levels compared to the PBS control were selected for further studies.

TABLE 55 Effect of antisense oligonucleotide treatment on BUN (mg/dL) in CD1 mice 2 weeks 4 weeks 6 weeks PBS 30 n.d. n.d. ISIS 416825 29 35 31 ISIS 416826 24 34 27 ISIS 416838 25 38 30 ISIS 416850 25 30 23 ISIS 416858 21 29 19 ISIS 416864 22 31 28 ISIS 416925 21 30 17 ISIS 416999 22 27 22 ISIS 417002 19 23 19 ISIS 416892 19 28 23 ISIS 417003 23 26 24 n.d. = no data

TABLE 56 Effect of antisense oligonucleotide treatment on creatinine (mg/dL) in CD1 mice 2 weeks 4 weeks 6 weeks PBS 0.14 n.d. n.d. ISIS 416825 0.14 0.21 0.17 ISIS 416826 0.15 0.20 0.15 ISIS 416838 0.09 0.27 0.14 ISIS 416850 0.13 0.22 0.19 ISIS 416858 0.13 0.23 0.10 ISIS 416864 0.11 0.22 0.16 ISIS 416925 0.12 0.25 0.13 ISIS 416999 0.07 0.18 0.13 ISIS 417002 0.06 0.16 0.10 ISIS 416892 0.11 0.20 0.17 ISIS 417003 0.17 0.24 0.18 n.d. = no data

Hematology Assays

Blood obtained from all mice groups were sent to Antech Diagnostics for hematocrit (HCT), mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH), and mean corpuscular hemoglobin concentration (MCHC) measurements and analyses, as well as measurements of the various blood cells, such as WBC (neutrophils, lymphocytes, and monocytes), RBC, and platelets, and total hemoglobin content. The results are presented in Tables 57-67. Percentages given in the tables indicate the percent of total blood cell count. Those antisense oligonucleotides which did not affect a decrease in platelet count of more than 50% and/or an increase in monocyte count of more than three-fold were selected for further studies.

TABLE 57 Effect of antisense oligonucleotide treatment on HCT (%) in CD1 mice 2 weeks 4 weeks 6 weeks PBS 50 n.d. n.d. ISIS 416825 49 46 40 ISIS 416826 47 41 37 ISIS 416838 42 44 39 ISIS 416850 44 44 38 ISIS 416858 50 45 46 ISIS 416864 50 45 42 ISIS 416925 51 47 47 ISIS 416999 51 42 40 ISIS 417002 44 44 51 ISIS 416892 48 42 45 ISIS 417003 48 41 43 n.d. = no data

TABLE 58 Effect of antisense oligonucleotide treatment on MCV (fL) in CD1 mice 2 weeks 4 weeks 6 weeks PBS 61 n.d. n.d. ISIS 416825 58 53 51 ISIS 416826 56 52 53 ISIS 416838 56 54 48 ISIS 416850 57 51 50 ISIS 416858 59 51 50 ISIS 416864 57 52 51 ISIS 416925 61 52 47 ISIS 416999 60 49 48 ISIS 417002 61 50 52 ISIS 416892 59 49 53 ISIS 417003 60 48 45 n.d. = no data

TABLE 59 Effect of antisense oligonucleotide treatment on MCH (pg) in CD1 mice ISIS No. 2 weeks 4 weeks 6 weeks PBS 18 n.d. n.d. ISIS 416825 17 16 15 ISIS 416826 17 16 16 ISIS 416838 17 17 15 ISIS 416850 17 16 15 ISIS 416858 17 16 15 ISIS 416864 18 16 16 ISIS 416925 17 16 15 ISIS 416999 17 16 15 ISIS 417002 17 16 16 ISIS 416892 18 16 16 ISIS 417003 17 16 16 n.d. = no data

TABLE 60 Effect of antisense oligonucleotide treatment on MCHC (%) in CD1 mice 2 weeks 4 weeks 6 weeks PBS 30 n.d. n.d. ISIS 416825 29 31 31 ISIS 416826 29 31 30 ISIS 416838 30 31 32 ISIS 416850 30 31 31 ISIS 416858 30 32 31 ISIS 416864 31 31 31 ISIS 416925 30 32 32 ISIS 416999 27 32 31 ISIS 417002 29 32 31 ISIS 416892 30 32 30 ISIS 417003 29 32 33 n.d. = no data

TABLE 61 Effect of antisense oligonucleotide treatment on WBC count (cells/nL) in CD1 mice 2 weeks 4 weeks 6 weeks PBS 6 n.d. n.d. ISIS 416825 8 8 6 ISIS 416826 5 6 8 ISIS 416838 4 6 5 ISIS 416850 4 5 5 ISIS 416858 6 7 4 ISIS 416864 7 6 5 ISIS 416925 6 6 11 ISIS 416999 4 9 7 ISIS 417002 8 8 16 ISIS 416892 5 8 9 ISIS 417003 7 9 10 n.d. = no data

TABLE 62 Effect of antisense oligonucleotide treatment on RBC count (cells/pL) in CD1 mice 2 weeks 4 weeks 6 weeks PBS 8 n.d. n.d. ISIS 416825 9 9 8 ISIS 416826 8 8 7 ISIS 416838 8 8 8 ISIS 416850 8 9 8 ISIS 416858 9 9 9 ISIS 416864 9 9 8 ISIS 416925 9 9 10 ISIS 416999 9 9 8 ISIS 417002 9 9 10 ISIS 416892 7 9 9 ISIS 417003 8 9 10 n.d. = no data

TABLE 63 Effect of antisense oligonucleotide treatment on neutrophil count (%) in CD1 mice 2 weeks 4 weeks 6 weeks PBS 16 n.d. n.d. ISIS 416825 15 43 23 ISIS 416826 26 33 23 ISIS 416838 19 33 31 ISIS 416850 15 21 16 ISIS 416858 14 24 27 ISIS 416864 13 27 20 ISIS 416925 12 39 33 ISIS 416999 12 25 22 ISIS 417002 14 31 36 ISIS 416892 19 43 28 ISIS 417003 10 39 24 n.d. = no data

TABLE 64 Effect of antisense oligonucleotide treatment on lymphocyte count (%) in CD1 mice 2 weeks 4 weeks 6 weeks PBS 81 n.d. n.d. ISIS 416825 82 53 71 ISIS 416826 70 61 67 ISIS 416838 76 64 60 ISIS 416850 82 73 76 ISIS 416858 83 73 65 ISIS 416864 84 71 74 ISIS 416925 86 58 57 ISIS 416999 86 72 69 ISIS 417002 83 64 51 ISIS 416892 79 52 64 ISIS 417003 86 54 66 n.d. = no data

TABLE 65 Effect of antisense oligonucleotide treatment on monocyte count (%) in CD1 mice 2 weeks 4 weeks 6 weeks PBS 3 n.d. n.d. ISIS 416825 2 5 4 ISIS 416826 3 5 8 ISIS 416838 2 2 6 ISIS 416850 3 6 6 ISIS 416858 2 3 7 ISIS 416864 2 2 5 ISIS 416925 2 4 8 ISIS 416999 2 4 8 ISIS 417002 3 4 12 ISIS 416892 3 6 7 ISIS 417003 2 6 8 n.d. = no data

TABLE 66 Effect of antisense oligonucleotide treatment on platelet count (cells/nL) in CD1 mice 2 weeks 4 weeks 6 weeks PBS 2126 n.d. n.d. ISIS 416825 1689 1229 942 ISIS 416826 1498 970 645 ISIS 416838 1376 1547 1229 ISIS 416850 1264 1302 1211 ISIS 416858 2480 1364 1371 ISIS 416864 1924 1556 933 ISIS 416925 1509 1359 1211 ISIS 416999 1621 1219 1057 ISIS 417002 1864 1245 1211 ISIS 416892 1687 636 1004 ISIS 417003 1309 773 922 n.d. = no data

TABLE 67 Effect of antisense oligonucleotide treatment on hemoglobin content (g/dL) in CD1 mice 2 weeks 4 weeks 6 weeks PBS 15.1 n.d. n.d. ISIS 416825 14.5 14.1 12.1 ISIS 416826 13.4 12.8 11.0 ISIS 416838 12.4 13.6 12.6 ISIS 416850 13.1 13.5 11.6 ISIS 416858 14.8 14.2 14.1 ISIS 416864 15.2 13.9 13.0 ISIS 416925 14.9 14.8 15.3 ISIS 416999 14.2 13.3 12.8 ISIS 417002 14.7 14.0 15.7 ISIS 416892 13.0 13.5 13.1 ISIS 417003 13.7 13.4 14.0 n.d. = no data

Example 34 Measurement of Half-Life of Antisense Oligonucleotide in CD1 Mice Liver

CD1 mice were treated with ISIS antisense oligonucleotides targeting human Factor 11 and the oligonucleotide half-life as well as the elapsed time for oligonucleotide degradation and elimination from the liver was evaluated.

Treatment

Groups of fifteen CD1 mice each were injected subcutaneously twice per week for 2 weeks with 50 mg/kg of ISIS 416825, ISIS 416826, ISIS 416838, ISIS 416850, ISIS 416858, ISIS 416864, ISIS 416892, ISIS 416925, ISIS 416999, ISIS 417002, or ISIS 417003. Five mice from each group were sacrificed 3 days, 28 days and 56 days following the final dose. Livers were harvested for analysis.

Measurement of Oligonucleotide Concentration

The concentration of the full-length oligonucleotide as well as the total oligonucleotide concentration (including the degraded form) was measured. The method used is a modification of previously published methods (Leeds et al., 1996; Geary et al., 1999) which consist of a phenol-chloroform (liquid-liquid) extraction followed by a solid phase extraction. An internal standard (ISIS 355868, a 27-mer 2′-O-methoxyethyl modified phosphorothioate oligonucleotide, GCGTTTGCTCTTCTTCTTGCGTTTTTT, designated herein as SEQ ID NO: 270) was added prior to extraction. Tissue sample concentrations were calculated using calibration curves, with a lower limit of quantitation (LLOQ) of approximately 1.14 μg/g. Half-lives were then calculated using WinNonlin software (PHARSIGHT).

The results are presented in Tables 68 and 69, expressed as μg/g liver tissue. The half-life of each oligonucleotide is presented in Table 70.

TABLE 68 Full-length oligonucleotide concentration (μg/g) in the liver of CD1 mice ISIS No. Motif day 3 day 28 day 56 416825 5-10-5 151 52 7 416826 5-10-5 186 48 8 416838 5-10-5 170 46 10 416850 5-10-5 238 93 51 416858 5-10-5 199 102 18 416864 5-10-5 146 38 25 416999 2-13-5 175 26 0 417002 2-13-5 119 24 1 417003 2-13-5 245 42 4 416925 3-14-3 167 39 5 416892 3-14-3 135 31 6

TABLE 69 Total oligonucleotide concentration (μg/g) in the liver of CD1 mice ISIS No. Motif day 3 day 28 day 56 416825 5-10-5 187 90 39 416826 5-10-5 212 61 12 416838 5-10-5 216 98 56 416850 5-10-5 295 157 143 416858 5-10-5 273 185 56 416864 5-10-5 216 86 112 416999 2-13-5 232 51 0 417002 2-13-5 206 36 1 417003 2-13-5 353 74 4 416925 3-14-3 280 72 8 416892 3-14-3 195 54 6

TABLE 70 Half-life of antisense oligonucleotides in the liver of CD1 mice Half-life ISIS No. Motif (days) 416825 5-10-5 16 416826 5-10-5 13 416838 5-10-5 13 416850 5-10-5 18 416858 5-10-5 26 416864 5-10-5 13 416999 2-13-5 9 417002 2-13-5 11 417003 2-13-5 10 416925 3-14-3 12 416892 3-14-3 12

Example 35 Tolerability of Antisense Oligonucleotides Targeting Human Factor 11 in Sprague-Dawley Rats

Sprague-Dawley rats were treated with ISIS antisense oligonucleotides targeting human Factor 11 and evaluated for changes in the levels of various metabolic markers.

Treatment

Groups of four Sprague Dawley rats each were injected subcutaneously twice per week for 6 weeks with 50 mg/kg of ISIS 416825, ISIS 416826, ISIS 416838, ISIS 416850, ISIS 416858, ISIS 416848, ISIS 416864, ISIS 416892, ISIS 416925, ISIS 416999, ISIS 417002, or ISIS 417003. A control group of four Sprague Dawley rats was injected subcutaneously with PBS twice per week for 6 weeks. Body weight measurements were taken before and throughout the treatment period. Urine samples were taken before the start of treatment. Three days after the last dose, urine samples were taken and the rats were sacrificed. Organ weights were measured and blood was collected for further analysis.

Body Weight and Organ Weight

Body weights of the rats were measured at the onset of the study and subsequently twice per week. The body weights are presented in Table 71 and are expressed as a percent change over the weights taken at the start of the study. Liver, spleen, and kidney weights were measured at the end of the study and are presented in Table 71 as a percent of the saline control normalized to body weight. Those antisense oligonucleotides which did not affect more than a six-fold increase in liver and spleen weight above the PBS control were selected for further studies.

TABLE 71 Percent change in organ weight of Sprague Dawley rats after antisense oligonucleotide treatment ISIS Body No. Liver Spleen Kidney weight 416825 +20 +245 +25 −18 416826 +81 +537 +44 −40 416838 +8 +212 −0.5 −23 416850 +23 +354 +47 −33 416858 +8 +187 +5 −21 416864 +16 +204 +16 −24 416925 +44 +371 +48 −32 416999 +51 +405 +71 −37 417002 +27 +446 +63 −29 416892 +38 +151 +32 −39 417003 +51 +522 +25 −40

Liver Function

To evaluate the effect of ISIS oligonucleotides on hepatic function, plasma concentrations of transaminases were measured using an automated clinical chemistry analyzer (Hitachi Olympus AU400e, Melville, N.Y.). Measurements of alanine transaminase (ALT) and aspartate transaminase (AST) are expressed in IU/L and the results are presented in Table 72. Those antisense oligonucleotides which did not affect an increase in ALT/AST levels above seven-fold of control levels were selected for further studies. Plasma levels of bilirubin and albumin were also measured with the same clinical analyzer and the results are also presented in Table 72, expressed in mg/dL. Those antisense oligonucleotides which did not affect an increase in levels of bilirubin more than two-fold of the control levels by antisense oligonucleotide treatment were selected for further studies.

TABLE 72 Effect of antisense oligonucleotide treatment on metabolic markers in the liver of Sprague-Dawley rats ALT AST Bilirubin Albumin (IU/L) (IU/L) (mg/dL) (mg/dL) PBS 9 5 20 2 ISIS 416825 89 17 4 2 ISIS 416826 611 104 115 6 ISIS 416838 5 2 4 2 ISIS 416850 80 5 1 4 ISIS 416858 13 4 4 2 ISIS 416864 471 68 3 4 ISIS 416925 102 20 13 5 ISIS 416999 92 28 54 5 ISIS 417002 44 11 12 3 ISIS 416892 113 183 1 8 ISIS 417003 138 23 50 6

Kidney Function

To evaluate the effect of kidney function, plasma concentrations of blood urea nitrogen (BUN) and creatinine were measured using an automated clinical chemistry analyzer (Hitachi Olympus AU400e, Melville, N.Y.). Results are presented in Table 73, expressed in mg/dL. Those antisense oligonucleotides which did not affect more than a two-fold increase in BUN levels compared to the PBS control were selected for further studies. The ratio of urine protein to creatinine in total urine samples was also calculated before and after antisense oligonucleotide treatment and is presented in Table 74. Those antisense oligonucleotides which did not affect more than a five-fold increase in urine protein/creatinine ratios compared to the PBS control were selected for further studies.

TABLE 73 Effect of antisense oligonucleotide treatment on metabolic markers in the kidney of Sprague-Dawley rats BUN Creatinine PBS 4 8 ISIS 416825 7 17 ISIS 416826 25 6 ISIS 416838 4 5 ISIS 416850 5 7 ISIS 416858 8 4 ISIS 416864 5 6 ISIS 416925 7 5 ISIS 416999 2 4 ISIS 417002 11 1 ISIS 416892 188 1 ISIS 417003 9 9

TABLE 74 Effect of antisense oligonucleotide treatment on urine protein/creatinine ratio in Sprague Dawley rats Before After PBS 1.2 1.3 416825 1.1 5.4 416826 1.0 11.4 416838 1.2 3.7 416850 1.0 4.0 416858 0.9 4.4 416864 1.2 4.0 416925 1.0 4.3 416999 1.3 9.1 417002 1.0 2.4 416892 0.8 21.3 417003 0.9 4.8

Hematology Assays

Blood obtained from all rat groups were sent to Antech Diagnostics for hematocrit (HCT), mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCV), and mean corpuscular hemoglobin concentration (MCHC) measurements and analyses, as well as measurements of various blood cells, such as WBC (neutrophils, lymphocytes and monocytes), RBC, and platelets as well as hemoglobin content. The results are presented in Tables 75 and 76. Those antisense oligonucleotides which did not, affect a decrease in platelet count of more than 50% and an increase in monocyte count of more than three-fold were selected for further studies.

TABLE 75 Effect of antisense oligonucleotide treatment on blood cell count in Sprague-Dawley rats WBC RBC Neutrophils Lymphocytes Monocytes Platelets (/nL) (/pL) (%) (%) (%) (10³/μL) PBS 21 6 37 7 26 18 ISIS 416825 22 2 25 3 15 6 ISIS 416826 7 5 30 5 7 11 ISIS 416838 13 4 17 3 6 27 ISIS 416850 16 7 48 8 11 26 ISIS 416858 28 2 20 3 10 19 ISIS 416864 15 4 26 2 29 12 ISIS 416925 24 6 20 4 23 8 ISIS 416999 12 5 23 3 20 12 ISIS 417002 23 5 22 4 25 7 ISIS 416892 68 12 92 18 58 66 ISIS 417003 83 11 17 3 6 19

TABLE 76 Effect of antisense oligonucleotide treatment on hematologic factors (% control) in Sprague-Dawley rats Hemoglobin HCT MCV MCH MCHC (g/dL) (%) (fL) (pg) (%) PBS 6 4 6 2 4 ISIS 416825 2 2 4 2 4 ISIS 416826 7 7 6 3 4 ISIS 416838 2 5 4 2 5 ISIS 416850 4 5 3 4 2 ISIS 416858 2 3 2 2 1 ISIS 416864 4 2 4 2 4 ISIS 416925 6 8 5 2 4 ISIS 416999 6 5 2 3 1 ISIS 417002 5 7 7 3 5 ISIS 416892 14 13 1 2 0 ISIS 417003 11 8 6 4 4

Example 36 Measurement of Half-Life of Antisense Oligonucleotide in Sprague-Dawley Rat Liver and Kidney

Sprague Dawley rats were treated with ISIS antisense oligonucleotides targeting human Factor 11 and the oligonucleotide half-life as well as the elapsed time for oligonucleotide degradation and elimination from the liver and kidney was evaluated.

Treatment

Groups of four Sprague Dawley rats each were injected subcutaneously twice a week for 2 weeks with 20 mg/kg of ISIS416825, ISIS 416826, ISIS 416838, ISIS 416850, ISIS 416858, ISIS 416864, ISIS 416892, ISIS 416925, ISIS 416999, ISIS 417002, or ISIS 417003. Three days after the last dose, the rats were sacrificed and livers and kidneys were collected for analysis.

Measurement of Oligonucleotide Concentration

The concentration of the full-length oligonucleotide as well as the total oligonucleotide concentration (including the degraded form) was measured. The method used is a modification of previously published methods (Leeds et al., 1996; Geary et al., 1999) which consist of a phenol-chloroform (liquid-liquid) extraction followed by a solid phase extraction. An internal standard (ISIS 355868, a 27-mer 2′-O-methoxyethyl modified phosphorothioate oligonucleotide, GCGTTTGCTCTTCTTCTTGCGTTTTTT, designated herein as SEQ ID NO: 270) was added prior to extraction. Tissue sample concentrations were calculated using calibration curves, with a lower limit of quantitation (LLOQ) of approximately 1.14 μg/g. The results are presented in Tables 77 and 78, expressed as μg/g liver or kidney tissue. Half-lives were then calculated using WinNonlin software (PHARSIGHT).

TABLE 77 Full-length oligonucleotide concentration (μg/g) in the liver and kidney of Sprague-Dawley rats ISIS No. Motif Kidney Liver 416825 5-10-5 632 236 416826 5-10-5 641 178 416838 5-10-5 439 171 416850 5-10-5 259 292 416858 5-10-5 575 255 416864 5-10-5 317 130 416999 2-13-5 358 267 417002 2-13-5 291 118 417003 2-13-5 355 199 416925 3-14-3 318 165 416892 3-14-3 351 215

TABLE 78 Total oligonucleotide concentration (μg/g) in the liver and kidney of Sprague-Dawley rats ISIS No. Motif Kidney Liver 416825 5-10-5 845 278 416826 5-10-5 775 214 416838 5-10-5 623 207 416850 5-10-5 352 346 416858 5-10-5 818 308 416864 5-10-5 516 209 416999 2-13-5 524 329 417002 2-13-5 490 183 417003 2-13-5 504 248 416925 3-14-3 642 267 416892 3-14-3 608 316

TABLE 79 Half-life (days) of ISIS oligonucleotides in the liver and kidney of Sprague-Dawley rats ISIS No. Motif Half-life 416825 5-10-5 16 416826 5-10-5 13 416838 5-10-5 13 416850 5-10-5 18 416858 5-10-5 26 416864 5-10-5 13 416999 2-13-5 9 417002 2-13-5 11 417003 2-13-5 10 416925 3-14-3 12 416892 3-14-3 12

Example 37 Tolerability of Antisense Oligonucleotides Targeting Human Factor 11 in CD1 Mice

CD1 mice were treated with ISIS antisense oligonucleotides targeting human Factor 11 and evaluated for changes in the levels of various metabolic markers.

Treatment

Groups of five CD1 mice each were injected subcutaneously twice per week for 6 weeks with 50 mg/kg of ISIS 412223, ISIS 412224, ISIS 412225, ISIS 413481, ISIS 413482, ISIS 416848, ISIS 416849, ISIS 416850, ISIS 416851, ISIS 416852, ISIS 416853, ISIS 416854, ISIS 416855, ISIS 416856, ISIS 416857, ISIS 416858, ISIS 416859, ISIS 416860, ISIS 416861, ISIS 416862, ISIS 416863, ISIS 416864, ISIS 416865, ISIS 416866, or ISIS 416867, or. A control group of ten CD1 mice was injected subcutaneously with PBS twice per week for 6 weeks. Body weight measurements were taken before and throughout the treatment period. Three days after the last dose, the mice were sacrificed, organ weights were measured, and blood was collected for further analysis.

Body Weight and Organ Weights

Body weight was measured at the onset of the study and subsequently twice per week. The body weights of the mice are presented in Table 80 and are expressed increase in grams over the PBS control weight taken before the start of treatment. Liver, spleen, and kidney weights were measured at the end of the study, and are also presented in Table 80 as percentage of the body weight. Those antisense oligonucleotides which did not affect more than six-fold increases in liver and spleen weight above the PBS control were selected for further studies.

TABLE 80 Change in body and organ weights of CD1 mice after antisense oligonucleotide treatment body Liver Kidney Spleen weight (%) (%) (%) (g) PBS 5 1.5 0.3 7 ISIS 416850 6 1.6 0.4 12 ISIS 416858 7 1.6 0.6 12 ISIS 416864 5 1.6 0.3 12 ISIS 412223 6 1.5 0.4 12 ISIS 412224 6 1.6 0.5 10 ISIS 412225 6 1.5 0.4 10 ISIS 413481 6 1.5 0.5 9 ISIS 413482 6 1.6 0.5 11 ISIS 416848 6 1.5 0.4 11 ISIS 416849 8 1.5 0.4 8 ISIS 416851 7 1.5 0.5 11 ISIS 416852 6 1.5 0.4 10 ISIS 416853 8 1.5 0.7 13 ISIS 416854 7 1.2 0.4 13 ISIS 416855 8 1.4 0.6 12 ISIS 416856 6 1.4 0.4 10 ISIS 416857 7 1.6 0.5 10 ISIS 416859 6 1.5 0.4 10 ISIS 416860 6 1.4 0.4 10 ISIS 416861 5 1.3 0.4 9 ISIS 416862 6 1.5 0.4 10 ISIS 416863 5 1.5 0.4 9 ISIS 416865 6 1.5 0.4 8 ISIS 416866 5 1.6 0.4 10 ISIS 416867 5 1.4 0.4 9

Liver Function

To evaluate the effect of ISIS oligonucleotides on hepatic function, plasma concentrations of transaminases were measured using an automated clinical chemistry analyzer (Hitachi Olympus AU400e, Melville, N.Y.). Measurements of alanine transaminase (ALT) and aspartate transaminase (AST) are expressed in IU/L and the results are presented in Table 81. Those antisense oligonucleotides which did not affect an increase in ALT/AST levels above seven-fold of control levels were selected for further studies. Plasma levels of bilirubin, cholesterol and albumin were also measured using the same clinical chemistry analyzer and are presented in Table 81 expressed in mg/dL. Those antisense oligonucleotides which did not affect an increase in levels of bilirubin more than two-fold of the control levels by antisense oligonucleotide treatment were selected for further studies.

TABLE 81 Effect of antisense oligonucleotide treatment on metabolic markers in the liver of CD1 mice ALT AST Bilirubin Albumin Cholesterol (IU/L) (IU/L) (mg/dL) (mg/dL) (mg/dL) PBS 32 68 0.25 3.7 135 ISIS 416850 75 99 0.21 3.5 142 ISIS 416858 640 547 0.28 4.4 181 ISIS 416864 36 67 0.19 2.6 152 ISIS 412223 60 125 0.20 3.0 117 ISIS 412224 214 183 0.19 3.4 114 ISIS 412225 40 69 0.23 3.3 128 ISIS 413481 85 143 0.18 3.2 153 ISIS 413482 54 77 0.24 3.0 138 ISIS 416848 153 153 0.19 3.1 151 ISIS 416849 1056 582 0.22 2.5 109 ISIS 416851 47 76 0.19 3.1 106 ISIS 416852 49 91 0.16 4.9 125 ISIS 416853 1023 1087 0.25 3.1 164 ISIS 416854 1613 1140 0.21 5.5 199 ISIS 416855 786 580 0.25 4.2 162 ISIS 416856 130 129 0.23 5.2 109 ISIS 416857 370 269 0.22 3.7 94 ISIS 416859 214 293 0.20 4.2 160 ISIS 416860 189 160 0.23 3.5 152 ISIS 416861 38 85 0.27 4.3 133 ISIS 416862 225 172 0.36 3.9 103 ISIS 416863 41 101 0.24 3.6 118 ISIS 416865 383 262 0.27 4.1 95 ISIS 416866 36 120 0.29 4.3 113 ISIS 416867 45 82 0.21 3.3 144

Kidney Function

To evaluate the effect of ISIS oligonucleotides on kidney function, plasma concentrations of blood urea nitrogen (BUN) were measured using an automated clinical chemistry analyzer and results are presented in Table 82 expressed in mg/dL. Those antisense oligonucleotides which did not affect more than a two-fold increase in BUN levels compared to the PBS control were selected for further studies.

TABLE 82 Effect of antisense oligonucleotide treatment on BUN levels (mg/dL) in the kidney of CD1 mice BUN PBS 22 ISIS 416850 24 ISIS 416858 23 ISIS 416864 24 ISIS 412223 28 ISIS 412224 29 ISIS 412225 23 ISIS 413481 23 ISIS 413482 27 ISIS 416848 23 ISIS 416849 23 ISIS 416851 21 ISIS 416852 21 ISIS 416853 22 ISIS 416854 27 ISIS 416855 23 ISIS 416856 21 ISIS 416857 17 ISIS 416859 18 ISIS 416860 25 ISIS 416861 23 ISIS 416862 21 ISIS 416863 22 ISIS 416865 20 ISIS 416866 22 ISIS 416867 20

Hematology Assays

Blood obtained from all the mice groups were sent to Antech Diagnostics for hematocrit (HCT) measurements, as well as measurements of various blood cells, such as WBC (neutrophils, lymphocytes, and monocytes), RBC, and platelets, as well as total hemoglobin content analysis. The results are presented in Tables 83 and 84. Those antisense oligonucleotides which did not affect a decrease in platelet count of more than 50% and an increase in monocyte count of more than three-fold were selected for further studies.

TABLE 83 Effect of antisense oligonucleotide treatment on hematologic factors in CD1 mice RBC Hemoglobin HCT WBC (10⁶/μL) (g/dL) (%) (10³/μL) PBS 10 15 51 7 ISIS 416850 10 15 49 5 ISIS 416858 9 14 50 8 ISIS 416864 10 15 52 5 ISIS 412223 9 15 48 7 ISIS 412224 10 15 50 9 ISIS 412225 9 15 50 7 ISIS 413481 9 13 45 7 ISIS 413482 10 15 50 8 ISIS 416848 9 14 47 7 ISIS 416849 9 14 48 9 ISIS 416851 9 14 47 6 ISIS 416852 9 14 49 5 ISIS 416853 11 17 56 8 ISIS 416854 9 13 43 12 ISIS 416855 9 14 50 6 ISIS 416856 9 14 47 5 ISIS 416857 10 15 53 6 ISIS 416859 10 15 49 6 ISIS 416860 10 15 51 7 ISIS 416861 9 14 48 7 ISIS 416862 9 14 49 6 ISIS 416863 9 14 48 7 ISIS 416865 9 14 50 7 ISIS 416866 9 15 51 6 ISIS 416867 10 14 47 8

TABLE 84 Effect of antisense oligonucleotide treatment on blood cell count in CD1 mice Neutrophil Lymphocyte Monocytes Platelets (cells/μL) (cells/μL) (cells/μL) (10³/μL) PBS 1023 6082 205 940 ISIS 416850 1144 4004 156 916 ISIS 416858 2229 5480 248 782 ISIS 416864 973 3921 141 750 ISIS 412223 1756 4599 200 862 ISIS 412224 2107 6284 195 647 ISIS 412225 1547 4969 293 574 ISIS 413481 1904 4329 204 841 ISIS 413482 1958 5584 275 818 ISIS 416848 1264 5268 180 953 ISIS 416849 1522 6967 253 744 ISIS 416851 1619 4162 194 984 ISIS 416852 1241 3646 189 903 ISIS 416853 2040 5184 225 801 ISIS 416854 2082 9375 455 1060 ISIS 416855 1443 4236 263 784 ISIS 416856 1292 3622 151 753 ISIS 416857 1334 3697 215 603 ISIS 416859 1561 4363 229 826 ISIS 416860 1291 4889 161 937 ISIS 416861 1122 5119 219 836 ISIS 416862 1118 4445 174 1007 ISIS 416863 1330 5617 226 1131 ISIS 416865 1227 5148 315 872 ISIS 416866 1201 4621 211 1045 ISIS 416867 1404 6078 188 1006

Example 38 Measurement of half-life of antisense oligonucleotide in CD1 mouse liver

Fifteen antisense oligonucleotides which had been evaluated in CD1 mice (Example 37) were further evaluated. CD1 mice were treated with ISIS antisense oligonucleotides and the oligonucleotide half-life as well the elapsed time for oligonucleotide degradation and elimination in the liver was evaluated.

Treatment

Groups of fifteen CD1 mice each were injected subcutaneously twice per week for 2 weeks with 50 mg/kg of ISIS 412223, ISIS 412225, ISIS 413481, ISIS 413482, ISIS 416851, ISIS 416852, ISIS 416856, ISIS 416860, ISIS 416861, ISIS 416863, ISIS 416866, ISIS 416867, ISIS 412224, ISIS 416848 or ISIS 416859. Five mice from each group were sacrificed 3 days, 28 days, and 56 days after the last dose, livers were collected for analysis.

Measurement of Oligonucleotide Concentration

The concentration of the full-length oligonucleotide was measured. The method used is a modification of previously published methods (Leeds et al., 1996; Geary et al., 1999) which consist of a phenol-chloroform (liquid-liquid) extraction followed by a solid phase extraction. An internal standard (ISIS 355868, a 27-mer 2′-O-methoxyethyl modified phosphorothioate oligonucleotide, GCGTTTGCTCTTCTTCTTGCGTTTTTT, designated herein as SEQ ID NO: 270) was added prior to extraction. Tissue sample concentrations were calculated using calibration curves, with a lower limit of quantitation (LLOQ) of approximately 1.14 μg/g. The results are presented in Table 85 expressed as μg/g liver tissue. The half-life of each oligonucleotide was also presented in Table 85.

TABLE 85 Full-length oligonucleotide concentration and half-life in the liver of CD1 mice Half-Life ISIS No Motif day 3 day 28 day 56 (days) 412223 5-10-5 276 127 52 21.9 412224 5-10-5 287 111 31 16.6 412225 5-10-5 279 91 47 20.7 413481 5-10-5 185 94 31 20.6 413482 5-10-5 262 95 40 19.5 416848 5-10-5 326 147 68 23.5 416851 5-10-5 319 147 68 23.8 416852 5-10-5 306 145 83 28.4 416856 5-10-5 313 115 46 19.2 416859 5-10-5 380 156 55 19.0 416860 5-10-5 216 96 36 20.6 416861 5-10-5 175 59 39 24.5 416863 5-10-5 311 101 48 19.8 416866 5-10-5 246 87 25 16.0 416867 5-10-5 246 87 35 18.9

Example 39 Tolerability of Antisense Oligonucleotides Targeting Human Factor 11 in Sprague-Dawley Rats

Fifteen antisense oligonucleotides which had been evaluated in CD1 mice (Example 37) were further evaluated in Sprague-Dawley rats for changes in the levels of various metabolic markers.

Treatment

Groups of four Sprague Dawley rats each were injected subcutaneously twice per week for 6 weeks with 50 mg/kg of ISIS 412223, ISIS 412224, ISIS 412225, ISIS 413481, ISIS 413482, ISIS 416848, ISIS 416851, ISIS 416852, ISIS 416856, ISIS 416859, ISIS 416860, ISIS 416861, ISIS 416863, ISIS 416866, or ISIS 416867. A control group of four Sprague Dawley rats was injected subcutaneously with PBS twice per week for 6 weeks. Body weight measurements were taken before and throughout the treatment period. Three days after the last dose, urine samples were collected and the rats were then sacrificed, organ weights were measured, and blood was collected for further analysis.

Body Weight and Organ Weights

The body weights of the rats were measured at the onset of the study and subsequently twice per week. The body weights are presented in Table 86 and are expressed as increase in grams over the PBS control weight taken before the start of treatment. Liver, spleen and kidney weights were measured at the end of the study, and are also presented in Table 86 as a percentage of the body weight. Those antisense oligonucleotides which did not affect more than six-fold increases in liver and spleen weight above the PBS control were selected for further studies.

TABLE 86 Change in body and organ weights of Sprague Dawley rats after antisense oligonucleotide treatment Body weight Liver Kidney Spleen (g) (%) (%) (%) PBS 179 4 0.9 0.2 ISIS 412223 126 5 1.0 0.5 ISIS 412224 165 5 1.0 0.5 ISIS 412225 184 4 1.0 0.5 ISIS 413481 147 5 0.9 0.3 ISIS 413482 158 5 1.0 0.6 ISIS 416848 117 5 1.1 0.8 ISIS 416851 169 5 0.9 0.3 ISIS 416852 152 5 1.0 0.4 ISIS 416856 156 5 1.0 0.4 ISIS 416859 128 4 1.0 0.4 ISIS 416860 123 5 1.0 0.5 ISIS 416861 182 5 0.9 0.3 ISIS 416863 197 5 1.0 0.4 ISIS 416866 171 5 1.0 0.5 ISIS 416867 129 5 1.0 0.5

Liver Function

To evaluate the effect of ISIS oligonucleotides on hepatic function, plasma concentrations of transaminases were measured using an automated clinical chemistry analyzer (Hitachi Olympus AU400e, Melville, N.Y.). Measurements of alanine transaminase (ALT) and aspartate transaminase (AST) are expressed in IU/L and the results are presented in Table 87. Those antisense oligonucleotides which did not affect an increase in ALT/AST levels above seven-fold of control levels were selected for further studies. Plasma levels of bilirubin and albumin were also measured using the same clinical chemistry analyzer and results are presented in Table 87 and expressed in mg/dL. Those antisense oligonucleotides which did not affect an increase in levels of bilirubin more than two-fold of the control levels by antisense oligonucleotide treatment were selected for further studies.

TABLE 87 Effect of antisense oligonucleotide treatment on metabolic markers in the liver of Sprague-Dawley rats ALT AST Bilirubin Albumin (IU/L) (IU/L) (mg/dL) (mg/dL) PBS 42 71 0.13 4 ISIS 412223 85 180 0.14 5 ISIS 412224 84 132 0.12 4 ISIS 412225 48 108 0.15 5 ISIS 413481 54 80 0.22 4 ISIS 413482 59 157 0.14 4 ISIS 416848 89 236 0.14 3 ISIS 416851 64 91 0.14 4 ISIS 416852 49 87 0.15 4 ISIS 416856 123 222 0.13 4 ISIS 416859 114 206 0.21 5 ISIS 416860 70 157 0.15 4 ISIS 416861 89 154 0.15 5 ISIS 416863 47 78 0.13 4 ISIS 416866 41 78 0.16 4 ISIS 416867 47 126 0.17 4

Kidney Function

To evaluate the effect of ISIS oligonucleotides on the kidney function, plasma concentrations of blood urea nitrogen (BUN) and creatinine were measured using an automated clinical chemistry analyzer (Hitachi Olympus AU400e, Melville, N.Y.). Results are presented in Table 88, expressed in mg/dL. Those antisense oligonucleotides which did not affect more than a two-fold increase in BUN levels compared to the PBS control were selected for further studies. The total urine protein and ratio of urine protein to creatinine in total urine samples after antisense oligonucleotide treatment was calculated and is also presented in Table 88. Those antisense oligonucleotides which did not affect more than a five-fold increase in urine protein/creatinine ratios compared to the PBS control were selected for further studies.

TABLE 88 Effect of antisense oligonucleotide treatment on metabolic markers in the kidney of Sprague-Dawley rats Total urine BUN Creatinine protein Urine protein/ (mg/dL) (mg/dL) (mg/dL) creatinine ratio PBS 19 38 60 1.7 ISIS 412223 24 46 224 4.6 ISIS 412224 24 44 171 3.8 ISIS 412225 23 58 209 4.0 ISIS 413481 26 45 148 3.6 ISIS 413482 23 34 157 4.8 ISIS 416848 26 64 231 3.9 ISIS 416851 24 70 286 4.0 ISIS 416852 25 60 189 3.0 ISIS 416856 23 48 128 2.7 ISIS 416859 24 44 144 3.3 ISIS 416860 23 58 242 4.6 ISIS 416861 22 39 205 5.1 ISIS 416863 29 73 269 3.8 ISIS 416866 22 85 486 6.2 ISIS 416867 22 70 217 3.1

Hematology Assays

Blood obtained from all rat groups were sent to Antech Diagnostics for hematocrit (HCT) measurements, as well as measurements of the various blood cells, such as WBC (neutrophils and lymphocytes), RBC, and platelets, and total hemoglobin content. The results are presented in Tables 89 and 90. Those antisense oligonucleotides which did not affect a decrease in platelet count of more than 50% and an increase in monocyte count of more than three-fold were selected for further studies.

TABLE 89 Effect of antisense oligonucleotide treatment on hematologic factors in Sprague-Dawley rats RBC Hemoglobin HCT WBC (10⁶/mL) (g/dL) (%) (10³/mL) PBS 6.9 13.2 42 9 ISIS 412223 7.2 13.1 41 20 ISIS 412224 7.4 13.4 42 20 ISIS 412225 7.4 13.4 42 15 ISIS 413481 7.5 14.2 43 14 ISIS 413482 7.1 13.2 40 13 ISIS 416848 6.0 11.1 35 17 ISIS 416851 7.4 13.7 42 11 ISIS 416852 7.2 13.4 42 13 ISIS 416856 7.7 14.1 43 19 ISIS 416859 7.8 14.0 45 16 ISIS 416860 7.8 14.1 45 17 ISIS 416861 7.7 14.6 45 15 ISIS 416863 7.6 14.1 45 17 ISIS 416866 7.8 14.0 44 20 ISIS 416867 7.8 14.0 45 14

TABLE 90 Effect of antisense oligonucleotide treatment on blood cell count in Sprague-Dawley rats Neutrophil Lymphocyte Platelets (/mL) (/mL) (10³/mL) PBS 988 7307 485 ISIS 412223 1826 16990 567 ISIS 412224 1865 16807 685 ISIS 412225 1499 13204 673 ISIS 413481 1046 12707 552 ISIS 413482 1125 11430 641 ISIS 416848 1874 14316 384 ISIS 416851 1001 9911 734 ISIS 416852 836 11956 632 ISIS 416856 3280 14328 740 ISIS 416859 1414 14323 853 ISIS 416860 1841 13986 669 ISIS 416861 1813 12865 1008 ISIS 416863 1720 14669 674 ISIS 416866 1916 16834 900 ISIS 416867 3044 10405 705

Example 40 Measurement of Half-Life of Antisense Oligonucleotide in the Liver and Kidney of Sprague-Dawley Rats

Sprague Dawley rats were treated with ISIS antisense oligonucleotides targeting human Factor 11 and the oligonucleotide half-life as well as the elapsed time for oligonucleotide degradation and elimination from the liver and kidney was evaluated.

Treatment

Groups of four Sprague Dawley rats each were injected subcutaneously twice per week for 2 weeks with 20 mg/kg of ISIS 412223, ISIS 412224, ISIS 412225, ISIS 413481, ISIS 413482, ISIS 416848, ISIS 416851, ISIS 416852, ISIS 416856, ISIS 416859, ISIS 416860, ISIS 416861, ISIS 416863, ISIS 416866, or ISIS 416867. Three days after the last dose, the rats were sacrificed, and livers and kidneys were harvested.

Measurement of Oligonucleotide Concentration

The concentration of the full-length oligonucleotide as well as the total oligonucleotide concentration (including the degraded form) was measured. The method used is a modification of previously published methods (Leeds et al., 1996; Geary et al., 1999) which consist of a phenol-chloroform (liquid-liquid) extraction followed by a solid phase extraction. An internal standard (ISIS 355868, a 27-mer 2′-O-methoxyethyl modified phosphorothioate oligonucleotide, GCGTTTGCTCTTCTTCTTGCGTTTTTT, designated herein as SEQ ID NO: 270) was added prior to extraction. Tissue sample concentrations were calculated using calibration curves, with a lower limit of quantitation (LLOQ) of approximately 1.14 μg/g. The results are presented in Tables 91 and 92, expressed as μg/g liver or kidney tissue. Half-lives were then calculated using WinNonlin software (PHARSIGHT).

TABLE 91 Full-length oligonucleotide concentration (μg/g) in the liver and kidney of Sprague-Dawley rats ISIS No Motif Kidney Liver 412223 5-10-5 551 97 412224 5-10-5 487 107 412225 5-10-5 202 119 413481 5-10-5 594 135 413482 5-10-5 241 95 416848 5-10-5 488 130 416851 5-10-5 264 193 416852 5-10-5 399 108 416856 5-10-5 378 84 416859 5-10-5 253 117 416860 5-10-5 247 94 416861 5-10-5 187 159 416863 5-10-5 239 82 416866 5-10-5 210 98 416867 5-10-5 201 112

TABLE 92 Total oligonucleotide concentration (μg/g) in the liver and kidney of Sprague-Dawley rats ISIS No Motif Kidney Liver 412223 5-10-5 395 86 412224 5-10-5 292 78 412225 5-10-5 189 117 413481 5-10-5 366 96 413482 5-10-5 217 91 416848 5-10-5 414 115 416851 5-10-5 204 178 416852 5-10-5 304 87 416856 5-10-5 313 80 416859 5-10-5 209 112 416860 5-10-5 151 76 416861 5-10-5 165 144 416863 5-10-5 203 79 416866 5-10-5 145 85 416867 5-10-5 157 98

TABLE 93 Half-life (days) of ISIS oligonucleotides in the liver and kidney of Sprague-Dawley rats ISIS No Motif Half-life 412223 5-10-5 22 412224 5-10-5 17 412225 5-10-5 21 413481 5-10-5 21 413482 5-10-5 20 416848 5-10-5 24 416851 5-10-5 24 416852 5-10-5 28 416856 5-10-5 19 416859 5-10-5 19 416860 5-10-5 21 416861 5-10-5 25 416863 5-10-5 20 416866 5-10-5 16 416867 5-10-5 19

Example 41 Tolerability of Antisense Oligonucleotides Targeting Human Factor 11 in CD1 Mice

ISIS oligonucleotides with 6-8-6 MOE and 5-8-5 MOE motifs targeting human Factor 11 were administered in CD1 mice evaluated for changes in the levels of various metabolic markers.

Treatment

Groups of five CD1 mice each were injected subcutaneously twice per week for 6 weeks with 50 mg/kg of ISIS 416850, ISIS 445498, ISIS 445503, ISIS 445504, ISIS 445505, ISIS 445509, ISIS 445513, ISIS 445522, ISIS 445530, ISIS 445531, or ISIS 445532. A control group of five CD1 mice was injected subcutaneously with PBS twice per week for 6 weeks. Body weight measurements were taken before and at the end of the treatment period. Three days after the last dose, the mice were sacrificed, organ weights were measured, and blood was collected for further analysis.

Body Weight and Organ Weight

The body weight changes in the mice are presented in Table 94 and are expressed increase in grams over the PBS control weight taken before the start of treatment. Liver, spleen and kidney weights were measured at the end of the study, and are also presented in Table 94 as percentage of the body weight. Those antisense oligonucleotides which did not affect more than six-fold increases in liver and spleen weight above the PBS control were selected for further studies.

TABLE 94 Change in body and organ weights of CD1 mice after antisense oligonucleotide treatment Body weight Liver Kidney Spleen (g) (%) (%) (%) PBS 10 5 1.6 0.3 ISIS 416850 11 6 1.5 0.4 ISIS 445498 10 6 1.6 0.5 ISIS 445503 9 8 1.4 0.6 ISIS 445504 11 6 1.6 0.4 ISIS 445505 12 6 1.5 0.5 ISIS 445509 10 6 1.6 0.5 ISIS 445513 9 5 1.6 0.4 ISIS 445522 11 6 1.7 0.4 ISIS 445530 11 6 1.5 0.5 ISIS 445531 10 6 1.5 0.5 ISIS 445532 10 6 1.6 0.4

Liver Function

To evaluate the effect of ISIS oligonucleotides on hepatic function, plasma concentrations of transaminases were measured using an automated clinical chemistry analyzer (Hitachi Olympus AU400e, Melville, N.Y.). Measurements of alanine transaminase (ALT) and aspartate transaminase (AST) are expressed in IU/L and the results are presented in Table 95. Those antisense oligonucleotides which did not affect an increase in ALT/AST levels above seven-fold of control levels were selected for further studies. Plasma levels of bilirubin and albumin were also measured and results are also presented in Table 95 and expressed in mg/dL. Those antisense oligonucleotides which did not affect an increase in levels of bilirubin more than two-fold of the control levels by antisense oligonucleotide treatment were selected for further studies.

TABLE 95 Effect of antisense oligonucleotide treatment on metabolic markers in the liver of CD1 mice ALT AST Bilirubin Albumin (IU/L) (IU/L) (mg/dL) (mg/dL) PBS 34 49 0.23 3.6 ISIS 416850 90 115 0.20 3.2 ISIS 445498 66 102 0.24 3.4 ISIS 445503 1314 852 0.28 3.4 ISIS 445504 71 107 0.17 3.4 ISIS 445505 116 153 0.18 3.2 ISIS 445509 80 117 0.17 3.1 ISIS 445513 37 84 0.22 3.1 ISIS 445522 51 110 0.19 3.4 ISIS 445530 104 136 0.18 3.2 ISIS 445531 60 127 0.16 3.2 ISIS 445532 395 360 0.20 2.9

Kidney Function

To evaluate the effect of ISIS oligonucleotides on kidney function, plasma concentrations of blood urea nitrogen (BUN) were measured using an automated clinical chemistry analyzer (Hitachi Olympus AU400e, Melville, N.Y.). Results are presented in Table 96, expressed in mg/dL. Those antisense oligonucleotides which did not affect more than a two-fold increase in BUN levels compared to the PBS control were selected for further studies.

TABLE 96 Effect of antisense oligonucleotide treatment on BUN levels (mg/dL) in the kidney of CD1 mice BUN PBS 29 ISIS 416850 28 ISIS 445498 28 ISIS 445503 29 ISIS 445504 29 ISIS 445505 29 ISIS 445509 29 ISIS 445513 27 ISIS 445522 28 ISIS 445530 26 ISIS 445531 27 ISIS 445532 23

Hematology Assays

Blood obtained from all mice groups were sent to Antech Diagnostics for hematocrit (HCT) measurements, as well as measurements of the various blood cells, such as WBC (neutrophils and lymphocytes), RBC, and platelets, and total hemoglobin content. The results are presented in Tables 97 and 98. Those antisense oligonucleotides which did not affect a decrease in platelet count of more than 50% and an increase in monocyte count of more than three-fold were selected for further studies.

TABLE 97 Effect of antisense oligonucleotide treatment on hematologic factors in CD1 mice RBC Hemoglobin HCT WBC (10⁶/mL) (g/dL) (%) (10³/mL) PBS 9.6 15.0 51 6 ISIS 416850 9.8 14.8 50 6 ISIS 445498 9.4 13.9 47 5 ISIS 445503 9.2 13.6 46 8 ISIS 445504 9.6 14.7 49 5 ISIS 445505 9.6 14.6 49 5 ISIS 445509 10.2 15.3 51 5  ISIS 445513, 9.8 15.0 50 7 ISIS 445522 9.7 14.6 49 5 ISIS 445530 10.0 15.1 50 7 ISIS 445531 9.4 14.5 48 9 ISIS 445532 9.7 14.8 48 7

TABLE 98 Effect of antisense oligonucleotide treatment on blood cell count in CD1 mice Neutrophil Lymphocyte Platelets (/mL) (/mL) (10³/mL) PBS 1356 4166 749 ISIS 416850 1314 4710 614 ISIS 445498 1197 3241 802 ISIS 445503 1475 6436 309 ISIS 445504 959 3578 826 ISIS 445505 818 3447 725 ISIS 445509 1104 3758 1085 ISIS 445513 959 5523 942 ISIS 445522 698 3997 1005 ISIS 445530 930 5488 849 ISIS 445531 2341 6125 996 ISIS 445532 1116 5490 689

Example 42 Tolerability of Antisense Oligonucleotides Targeting Human Factor 11 in Sprague-Dawley Rats

Eight antisense oligonucleotides which had been evaluated in CD1 mice (Example 41) were further evaluated in Sprague-Dawley rats for changes in the levels of various metabolic markers.

Treatment

Groups of four Sprague Dawley rats each were injected subcutaneously twice per week for 6 weeks with 50 mg/kg of ISIS 445498, ISIS 445504, ISIS 445505, ISIS 445509, ISIS 445513, ISIS 445522, ISIS 445530, or ISIS 445531. A control group of Sprague Dawley rats was injected subcutaneously with PBS twice per week for 6 weeks. Body weight measurements were taken before and throughout the treatment period. Three days after the last dose, urine samples were collected and the rats were then sacrificed, organ weights were measured, and blood was collected for further analysis.

Body Weight and Organ Weight

The body weights of the rats were measured at the onset of the study and subsequently twice per week. The body weights are presented in Table 99 and are expressed as percent increase over the PBS control weight taken before the start of treatment. Liver, spleen and kidney weights were measured at the end of the study, and are also presented in Table 99 as a percentage of the body weight. Those antisense oligonucleotides which did not affect more than six-fold increases in liver and spleen weight above the PBS control were selected for further studies.

TABLE 99 Change in body and organ weights of Sprague Dawley rats after antisense oligonucleotide treatment (%) Body weight Liver Spleen Kidney ISIS 445498 −17 +26 +107 −10 ISIS 445504 −15 +22 +116 +6 ISIS 445505 −21 +12 +146 +2 ISIS 445509 −17 +16 +252 +3 ISIS 445513 −13 +25 +194 +15 ISIS 445522 −13 +26 +184 +19 ISIS 445530 −7 +24 +99 +4 ISIS 445531 −10 +17 +89 +4

Liver Function

To evaluate the effect of ISIS oligonucleotides on hepatic function, plasma concentrations of transaminases were measured using an automated clinical chemistry analyzer (Hitachi Olympus AU400e, Melville, N.Y.). Plasma concentrations of ALT (alanine transaminase) and AST (aspartate transaminase) were measured and the results are presented in Table 100 expressed in IU/L. Those antisense oligonucleotides which did not affect an increase in ALT/AST levels above seven-fold of control levels were selected for further studies. Plasma levels of bilirubin and albumin were also measured using the same clinical chemistry analyzer; results are presented in Table 100 and expressed in mg/dL. Those antisense oligonucleotides which did not affect an increase in levels of bilirubin more than two-fold of the control levels by antisense oligonucleotide treatment were selected for further studies.

TABLE 100 Effect of antisense oligonucleotide treatment on metabolic markers in the liver of Sprague-Dawley rats ALT AST Bilirubin Albumin (IU/L) (IU/L) (mg/dL) (mg/dL) PBS 102 36 0.13 3.7 ISIS 445498 417 124 0.14 3.7 ISIS 445504 206 86 0.11 3.5 ISIS 445505 356 243 0.15 3.6 ISIS 445509 676 291 0.14 3.5 ISIS 445513 214 91 0.15 3.5 ISIS 445522 240 138 0.47 3.6 ISIS 445530 116 56 0.11 3.7 ISIS 445531 272 137 0.12 3.7

Kidney Function

To evaluate the effect of ISIS oligonucleotides on kidney function, plasma concentrations of blood urea nitrogen (BUN) and creatinine were measured using an automated clinical chemistry analyzer (Hitachi Olympus AU400e, Melville, N.Y.). Results are presented in Table 101, expressed in mg/dL. Those antisense oligonucleotides which did not affect more than a two-fold increase in BUN levels compared to the PBS control were selected for further studies. The total urine protein and ratio of urine protein to creatinine in total urine samples after antisense oligonucleotide treatment was calculated and is also presented in Table 101. Those antisense oligonucleotides which did not affect more than a five-fold increase in urine protein/creatinine ratios compared to the PBS control were selected for further studies.

TABLE 101 Effect of antisense oligonucleotide treatment on metabolic markers in the kidney of Sprague-Dawley rats BUN Creatinine Urine protein/ (mg/dL) (mg/dL) creatinine ratio PBS 18 0.4 1.4 ISIS 445498 25 0.5 3.1 ISIS 445504 26 0.4 4.3 ISIS 445505 24 0.4 3.8 ISIS 445509 27 0.5 4.0 ISIS 445513 24 0.4 4.6 ISIS 445522 25 0.4 6.4 ISIS 445530 22 0.4 4.2 ISIS 445531 23 0.4 3.4

Hematology Assays

Blood obtained from all rat groups were sent to Antech Diagnostics for hematocrit (HCT) measurements, as well as measurements of the various blood cells, such as WBC (neutrophils, lymphocytes, and monocytes), RBC, and platelets, and total hemoglobin content. The results are presented in Tables 102 and 103. Those antisense oligonucleotides which did not affect a decrease in platelet count of more than 50% and an increase in monocyte count of more than three-fold were selected for further studies.

TABLE 102 Effect of antisense oligonucleotide treatment on hematologic factors in Sprague-Dawley rats RBC Hemoglobin HCT WBC (/pL) (g/dL) (%) (/nL) PBS 8.8 16.0 55 13 ISIS 445498 8.5 14.7 49 13 ISIS 445504 8.9 14.7 50 16 ISIS 445505 9.1 15.0 50 21 ISIS 445509 8.4 14.1 47 17 ISIS 445513 7.8 13.0 44 17 ISIS 445522 7.7 13.6 47 18 ISIS 445530 8.9 14.7 50 12 ISIS 445531 8.8 14.8 50 13

TABLE 103 Effect of antisense oligonucleotide treatment on blood cell count in Sprague-Dawley rats Neutrophil Lymphocyte Monocytes Platelets (%) (%) (%) (/nL) PBS 14 82 2.0 1007 ISIS 445498 9 89 2.0 1061 ISIS 445504 10 87 2.0 776 ISIS 445505 10 87 2.5 1089 ISIS 445509 11 84 3.8 1115 ISIS 445513 14 82 3.5 1051 ISIS 445522 13 84 2.8 1334 ISIS 445530 11 87 2.0 1249 ISIS 445531 10 86 2.8 1023

Example 43 Tolerability of Antisense Oligonucleotides Targeting Human Factor 11 in CD1 Mice

ISIS oligonucleotides with 4-8-4 MOE, 3-8-3 MOE, 2-10-2 MOE, 3-10-3 MOE, and 4-10-4 MOE motifs targeting human Factor 11 were administered in CD1 mice evaluated for changes in the levels of various metabolic markers.

Treatment

Groups of five CD1 mice each were injected subcutaneously twice per week for 6 weeks with 50 mg/kg of ISIS 449707, ISIS 449708, ISIS 449409, ISIS 449710, or ISIS 449711. A control group of five CD1 mice was injected subcutaneously with PBS twice per week for 6 weeks. Body weight measurements were taken before and at the end of the treatment period. Three days after the last dose, the mice were sacrificed, organ weights were measured, and blood was collected for further analysis.

Body Weight and Organ Weight

The body weights of the mice taken at the end of the study are presented in Table 104 and are expressed in grams. Liver, spleen and kidney weights were also measured at the end of the study and are also presented in Table 104 as percentage of the body weight. Those antisense oligonucleotides which did not affect more than six-fold increases in liver and spleen weight above the PBS control were selected for further studies.

TABLE 104 Change in body and organ weights of CD1 mice after antisense oligonucleotide treatment Body weight Liver Spleen Kidney (g) (%) (%) (%) PBS 39 — — — ISIS 449707 42 +11 +63 −5 ISIS 449708 40 +17 +66 0 ISIS 449709 40 +15 +62 −14 ISIS 449710 42 +6 +43 −7 ISIS 449711 42 +18 +63 −12

Liver Function

To evaluate the effect of ISIS oligonucleotides on hepatic function, plasma concentrations of transaminases were measured using an automated clinical chemistry analyzer (Hitachi Olympus AU400e, Melville, N.Y.). Plasma concentrations of ALT (alanine transaminase) and AST (aspartate transaminase) were measured and the results are presented in Table 105 expressed in IU/L. Those antisense oligonucleotides which did not affect an increase in ALT/AST levels above seven-fold of control levels were selected for further studies. Plasma levels of bilirubin and albumin were also measured using the same clinical chemistry analyzer and results are presented in Table 105 and expressed in mg/dL. Those antisense oligonucleotides which did not affect an increase in levels of bilirubin more than two-fold of the control levels by antisense oligonucleotide treatment were selected for further studies.

TABLE 105 Effect of antisense oligonucleotide treatment on metabolic markers in the liver of CD1 mice ALT AST Bilirubin Albumin (IU/L) (IU/L) (mg/dL) (mg/dL) PBS 39 52 0.22 3.2 ISIS 449707 41 62 0.19 2.3 ISIS 449708 66 103 0.17 2.8 ISIS 449709 62 83 0.18 2.8 ISIS 449710 43 95 0.18 2.8 ISIS 449711 52 83 0.22 2.8

Kidney Function

To evaluate the effect of ISIS oligonucleotides on kidney function, plasma concentrations of blood urea nitrogen (BUN) and creatinine were measured using an automated clinical chemistry analyzer (Hitachi Olympus AU400e, Melville, N.Y.). Results are presented in Table 106, expressed in mg/dL. Those antisense oligonucleotides which did not affect more than a two-fold increase in BUN levels compared to the PBS control were selected for further studies.

TABLE 106 Effect of antisense oligonucleotide treatment on metabolic markers (mg/dL) in the kidney of CD1 mice BUN Creatinine PBS 28 0.3 ISIS 449707 27 0.2 ISIS 449708 28 0.2 ISIS 449709 34 0.3 ISIS 449710 29 0.2 ISIS 449711 26 0.2

Hematology Assays

Blood obtained from all mice groups were sent to Antech Diagnostics for hematocrit (HCT), measurements, as well as measurements of the various blood cells, such as WBC (neutrophils, lymphocytes, and monocytes), RBC, and platelets, and total hemoglobin content. The results are presented in Tables 107 and 108. Those antisense oligonucleotides which did not affect a decrease in platelet count of more than 50% and an increase in monocyte count of more than three-fold were selected for further studies.

TABLE 107 Effect of antisense oligonucleotide treatment on hematologic factors in CD1 mice RBC Hemoglobin Hematocrit WBC (/pL) (g/dL) (%) (/nL) PBS 9.8 14.6 54 6 ISIS 449707 8.4 12.4 45 6 ISIS 449708 9.2 13.2 48 7 ISIS 449709 9.2 13.2 49 5 ISIS 449710 9.1 13.5 48 7 ISIS 449711 9.0 13.3 48 6

TABLE 108 Effect of antisense oligonucleotide treatment on blood cell count in CD1 mice Neutrophils Lymphocytes Monocytes Platelets (%) (%) (%) (/nL) PBS 15 80 3 1383 ISIS 449707 11 85 3 1386 ISIS 449708 17 77 5 1395 ISIS 449709 19 76 4 1447 ISIS 449710 15 81 3 1245 ISIS 449711 15 79 6 1225

Example 44 Tolerability of Antisense Oligonucleotides Targeting Human Factor 11 in Sprague-Dawley Rats

Five antisense oligonucleotides which had been evaluated in CD1 mice (Example 43) were further evaluated in Sprague-Dawley rats for changes in the levels of various metabolic markers.

Treatment

Groups of four Sprague Dawley rats each were injected subcutaneously twice per week for 6 weeks with 50 mg/kg of ISIS 449707, ISIS 449708, ISIS 449709, ISIS 449710, or ISIS 449711. A control group of four Sprague Dawley rats was injected subcutaneously with PBS twice per week for 6 weeks. Body weight measurements were taken before and throughout the treatment period.

Three days after the last dose, urine samples were collected and the rats were then sacrificed, organ weights were measured, and blood was collected for further analysis.

Body Weight and Organ Weight

The body weights of the rats were measured at the onset of the study and at the end of the study. The body weight changes are presented in Table 109 and are expressed as increase in grams over the PBS control weight taken before the start of treatment. Liver, spleen and kidney weights were measured at the end of the study, and are also presented in Table 109 as a percentage of the body weight. Those antisense oligonucleotides which did not affect more than six-fold increases in liver and spleen weight above the PBS control were selected for further studies.

TABLE 109 Change in body and organ weights of Sprague Dawley rats after antisense oligonucleotide treatment Body weight Liver Spleen Kidney (g) (%) (%) (%) PBS 478 — — — ISIS 449707 352 +41 +400 +80 ISIS 449708 382 +31 +259 +40 ISIS 449709 376 +8 +231 +19 ISIS 449710 344 +82 +302 +50 ISIS 449711 362 +52 +327 +72

Liver Function

To evaluate the impact of ISIS oligonucleotides on hepatic function, plasma concentrations of ALT and AST were measured using an automated clinical chemistry analyzer (Hitachi Olympus AU400e, Melville, N.Y.). Plasma concentrations of alanine transaminase (ALT) and aspartate transaminase (AST) were measured and the results are presented in Table 110 expressed in IU/L.

Those antisense oligonucleotides which did not affect an increase in ALT/AST levels above seven-fold of control levels were selected for further studies. Plasma levels of bilirubin and albumin were also measured and results are presented in Table 110 and expressed in mg/dL. Those antisense oligonucleotides which did not affect an increase in levels of bilirubin more than two-fold of the control levels by antisense oligonucleotide treatment were selected for further studies.

TABLE 110 Effect of antisense oligonucleotide treatment on metabolic markers in the liver of Sprague-Dawley rats ALT AST Bilirubin Albumin (IU/L) (IU/L) (mg/dL) (mg/dL) PBS 41 107 0.1 3.4 ISIS 449707 61 199 0.2 3.1 ISIS 449708 25 90 0.1 3.2 ISIS 449709 63 126 0.2 3.1 ISIS 449710 36 211 0.1 2.9 ISIS 449711 32 163 0.1 2.9

Kidney Function

To evaluate the impact of ISIS oligonucleotides on kidney function, plasma concentrations of BUN and creatinine were measured using an automated clinical chemistry analyzer (Hitachi Olympus AU400e, Melville, N.Y.). Results are presented in Table 111, expressed in mg/dL. Those antisense oligonucleotides which did not affect more than a two-fold increase in BUN levels compared to the PBS control were selected for further studies. The total urine protein and ratio of urine protein to creatinine in total urine samples after antisense oligonucleotide treatment was calculated and is also presented in Table 111. Those antisense oligonucleotides which did not affect more than a five-fold increase in urine protein/creatinine ratios compared to the PBS control were selected for further studies.

TABLE 111 Effect of antisense oligonucleotide treatment on metabolic markers in the kidney of Sprague-Dawley rats BUN Creatinine Urine protein/ (mg/dL) (mg/dL) creatinine ratio PBS 22 0.4 1.5 ISIS 449707 24 0.4 3.2 ISIS 449708 24 0.4 5.7 ISIS 449709 24 0.4 3.4 ISIS 449710 29 0.3 5.9 ISIS 449711 28 0.4 7.3

Hematology Assays

Blood obtained from all rat groups were sent to Antech Diagnostics for hematocrit (HCT) measurements, as well as measurements of the various blood cells, such as WBC (neutrophils, lymphocytes, and monocytes), RBC, and platelets, and total hemoglobin content. The results are presented in Tables 112 and 113. Those antisense oligonucleotides which did not affect a decrease in platelet count of more than 50% and an increase in monocyte count of more than three-fold were selected for further studies.

TABLE 112 Effect of antisense oligonucleotide treatment on hematologic factors in Sprague-Dawley rats RBC Hemoglobin Hematocrit WBC (/pL) (g/dL) (%) (/nL) PBS 8.2 15.1 50 16 ISIS 449707 6.0 12.0 40 20 ISIS 449708 6.6 12.2 40 22 ISIS 449709 6.9 12.6 41 14 ISIS 449710 6.3 12.5 41 13 ISIS 449711 6.4 12.6 43 13

TABLE 113 Effect of antisense oligonucleotide treatment on blood cell count in Sprague-Dawley rats Neutrophils Lymphocytes Monocytes Platelets (%) (%) (%) (/nL) PBS 12 84 2 1004 ISIS 449707 6 91 2 722 ISIS 449708 6 92 2 925 ISIS 449709 5 91 3 631 ISIS 449710 6 91 2 509 ISIS 449711 7 90 2 919

Example 45 Dose-Dependent Pharmacologic Effect of Antisense Oligonucleotides Targeting Human Factor 11 in Cynomolgus Monkeys

Several antisense oligonucleotides were tested in cynomolgus monkeys to determine the pharmacologic effects of the oligonucleotides on Factor 11 activity, anticoagulation and bleeding times, liver and kidney distributions, and tolerability. All the ISIS oligonucleotides used in this study target human Factor 11 mRNA and are also fully cross-reactive with the rhesus monkey gene sequence (see Table 44). It is expected that the rhesus monkey ISIS oligonucleotides are fully cross-reactive with the cynomolgus monkey gene sequence as well. At the time the study was undertaken, the cynomolgus monkey genomic sequence was not available in the National Center for Biotechnology Information (NCBI) database; therefore, cross-reactivity with the cynomolgus monkey gene sequence could not be confirmed.

Treatment

Groups, each consisting of two male and three female monkeys, were injected subcutaneously with ISIS 416838, ISIS 416850, ISIS 416858, ISIS 416864, or ISIS 417002 in escalating doses. Antisense oligonucleotide was administered to the monkeys at 5 mg/kg three times per a week for week 1; 5 mg/kg twice per week for weeks 2 and 3; 10 mg/kg three times per week for week 4; 10 mg/kg twice per week for weeks 5 and 6; 25 mg/kg three times per week for week 7; and 25 mg/kg twice per week for weeks 8, 9, 10, 11, and 12. One control group, consisting of two male and three female monkeys, was injected subcutaneously with PBS according to the same dosing regimen. An additional experimental group, consisting of two male and three female monkeys, was injected subcutaneously with ISIS 416850 in a chronic, lower dose regimen. Antisense oligonucleotide was administered to the monkeys at 5 mg/kg three times per week for week 1; 5 mg/kg twice per week for week 2 and 3; 10 mg/kg three times per week for week 4; and 10 mg/kg twice per week for weeks 5 to 12. Body weights were measured weekly. Blood samples were collected 14 days and 5 days before the start of treatment and subsequently once per week for Factor 11 protein activity analysis in plasma, fibrinogen measurement, PT and aPTT measurements, bleeding times, and measurement of various hematologic factors. On day 85, the monkeys were euthanized by exsanguination while under deep anesthesia, and organs harvested for further analysis.

RNA Analysis

On day 85, RNA was extracted from liver tissue for real-time PCR analysis of Factor 11 using primer probe set LTS00301 (forward primer sequence ACACGCATTAAAAAGAGCAAAGC, designated herein as SEQ ID NO 271; reverse primer sequence CAGTGTCATGGTAAAATGAAGAATGG, designated herein as SEQ ID NO: 272; and probe sequence TGCAGGCACAGCATCCCAGTGTTCTX, designated herein as SEQ ID NO. 273). Results are presented as percent inhibition of Factor 11, relative to PBS control. As shown in Table 114, treatment with ISIS oligonucleotides resulted in significant reduction of Factor 11 mRNA in comparison to the PBS control.

TABLE 114 Inhibition of Factor 11 mRNA in the cynomolgus monkey liver relative to the PBS control ISIS No % inhibition 416838 37 416850 84 416858 90 416864 44 417002 57

Protein Analysis

Plasma samples from all monkey groups taken on different days were analyzed by a sandwich-style ELISA assay (Affinity Biologicals Inc.) using an affinity-purified polyclonal anti-Factor 11 antibody as the capture antibody and a peroxidase-conjugated polyclonal anti-Factor 11 antibody as the detecting antibody. Monkey plasma was diluted 1:50 for the assay. Peroxidase activity was expressed by incubation with the substrate o-phenylenediamine. The color produced was quantified using a microplate reader at 490 nm and was considered to be proportional to the concentration of Factor 11 in the samples.

The results are presented in Table 115, expressed as percentage reduction relative to that of the PBS control. Treatment with ISIS 416850 and ISIS 416858 resulted in a time-dependent decrease in protein levels.

TABLE 115 Inhibition of Factor 11 protein in the cynomolgus monkey liver relative to the PBS control ISIS ISIS ISIS ISIS ISIS ISIS Days 416838 416850 416858 416864 417002 416850* −14 0 0 0 0 0 0 −5 0 0 0 5 0 1 8 3 8 6 7 0 6 15 4 4 16 9 4 13 22 5 11 23 7 2 12 29 8 15 28 10 8 20 36 11 17 35 9 8 22 43 5 23 39 9 9 24 50 8 42 49 10 13 30 57 10 49 60 7 24 34 64 11 55 68 5 26 37 71 12 57 71 10 30 41 78 10 63 73 9 22 42 85 10 64 78 8 23 34 PT and aPTT Assay

Blood samples were collected in tubes containing sodium citrate. PT and aPTT were determined in duplicate with an ACL 9000 coagulation instrument (Instrumentation Laboratory, Italy). The results were interpolated on a standard curve of serial dilutions citrated control monkey plasma tested to give a reported result in percent normal.

Prothrombin Time (PT) and Activated Partial Thromboplastin Time (aPTT) were measured using platelet poor plasma (PPP) from monkeys treated with ISIS oligonucleotides. PT and aPTT values are provided in Tables 116 and 117 and are reported as International Normalized Ratio (INR) values. INR values for PT and aPTT were determined by dividing the PT or aPTT value for each experimental group by the PT or aPTT for the PBS treated group. This ratio was then raised to the power of the International Sensitivity Index (ISI) of the tissue factor used. The ISIS oligonucleotide, ISIS 416850, given with the chronic dose regimen is distinguished from the other oligonucleotides with an asterisk (*).

As shown in Table 116, PT was not significantly prolonged in monkeys treated with ISIS oligonucleotides either in the escalating dose regimen or the chronic dose regimen. However, aPTT was prolonged in a dose-dependent manner, as presented in Table 117. These data suggest that antisense reduction of Factor 11 affects the contact activation pathway, but not the extrinsic pathway of blood coagulation. Therefore, antisense reduction of Factor 11 is useful for inhibiting the formation of a thrombus or clot in response to an abnormal vessel wall, but not in response to tissue injury.

TABLE 116 Effect of ISIS antisense oligonucleotides on PT ratio in cynomolgus monkeys ISIS ISIS ISIS ISIS ISIS ISIS day 416838 416850 416858 416864 417002 416850* −14 1.00 1.00 1.00 1.00 1.00 1.00 −5 1.00 1.00 1.00 1.00 1.00 1.00 8 1.03 1.00 1.05 1.02 1.02 1.03 15 1.03 1.02 1.07 1.07 1.04 1.06 22 1.07 1.02 1.06 1.03 1.04 1.06 29 1.03 1.03 1.08 1.06 1.01 1.00 36 1.05 1.02 1.07 1.06 1.05 1.06 43 1.03 1.01 1.08 1.04 1.03 1.02 50 1.02 1.02 1.03 1.01 0.99 0.98 57 1.04 1.04 1.09 1.08 1.03 n.d. 64 1.04 1.03 1.09 1.10 1.03 n.d. 71 1.02 1.03 1.07 1.07 0.99 n.d. 78 1.04 1.05 1.10 1.08 1.02 n.d. 85 1.05 1.04 1.07 1.13 1.02 n.d. n.d. = no data

TABLE 117 Effect of ISIS antisense oligonucleotides on aPTT ratio in cynomolgus monkeys ISIS ISIS ISIS ISIS ISIS ISIS day 416838 416850 416858 416864 417002 416850* −14 1.00 1.00 1.00 1.00 1.00 1.00 −5 1.00 1.00 1.00 1.00 1.00 1.00 8 1.07 1.05 1.03 1.05 1.05 1.12 15 1.05 1.05 1.07 1.03 1.03 1.07 22 1.20 1.13 1.18 1.11 1.16 1.21 29 1.19 1.13 1.20 1.13 1.11 1.26 36 1.20 1.26 1.36 1.19 1.18 1.34 43 1.18 1.17 1.28 1.07 1.06 1.22 50 1.25 1.68 1.55 1.26 1.18 1.35 57 1.21 1.59 1.59 1.19 1.22 n.d. 64 1.18 1.64 1.60 1.12 1.11 n.d. 71 1.15 1.76 1.70 1.18 1.16 n.d. 78 1.19 1.88 1.79 1.18 1.18 n.d. 85 1.22 1.99 1.76 1.25 1.20 n.d. n.d. = no data

Protein Activity Analysis

Blood samples were collected at various time points and Factor 11 proenzyme was measured using a F11 assay based on clotting time. Clotting times were determined in duplicate with a ST4 semi-automated coagulation instrument (Diagnostica Stago, N.J.). Thirty μl of citrated sample plasma diluted 1/20 in HEPES-NaCl buffer with BSA was incubated with 30 μl aPTT reagent (Automated aPTT, Organon Technika, N.C.) and 30 μl of citrated plasma deficient of Factor 11 (George King Bio-Medical Inc.) at 37° C. for 5 min, followed by the addition of 30 μl of 25 mM CaCl₂ to initiate clotting. Results were interpolated on a standard curve of serially diluted citrated control plasma.

Results are presented in Table 118 as percent inhibition of Factor 11 activity, relative to PBS control. The ISIS oligonucleotide, ISIS 416850, given with the chronic dose regimen is distinguished from the other oligonucleotides with an asterisk (*).

TABLE 118 Inhibition of Factor 11 protein by ISIS antisense oligonucleotides given in escalating dose/chronic dose regimen in cynomolgus monkeys Days before/after ISIS ISIS ISIS ISIS ISIS ISIS treatment 416838 416850 416858 416864 417002 416850* −14 0 0 0 0 0 0 −5 0 0 0 5 0 1 8 3 8 6 7 0 6 15 4 4 16 9 4 13 22 5 11 23 7 2 12 29 8 15 28 10 8 20 36 11 17 35 9 8 24 43 5 23 39 9 9 24 50 8 42 49 10 13 30 57 10 49 60 7 24 n.d. 64 11 55 68 5 26 n.d. 71 12 57 71 10 30 n.d. 78 10 63 73 9 22 n.d. 85 10 64 78 8 23 n.d. n.d. = no data

Fibrinogen Assay

Nine parts of fresh monkey plasma was collected into one part of trisodium citrate. The samples were evaluated of fibrinogen content using an ACL 9000 coagulation instrument (Instrumentation Laboratory, Italy). Results are presented in Table 119 expressed in mg/dL. The ISIS oligonucleotide, ISIS 416850, given with the chronic dose regimen is distinguished from the other oligonucleotides with an asterisk (*).

TABLE 119 Effect of ISIS antisense oligonucleotides on fibrinogen levels in cynomolgus monkeys Days before/after ISIS ISIS ISIS ISIS ISIS treatment PBS ISIS 416838 416850 416858 416864 417002 416850* −14 296 251 310 277 300 291 274 −5 246 205 261 246 243 222 227 8 245 209 281 246 227 221 232 15 207 198 270 219 210 195 174 22 219 183 243 222 184 199 192 29 231 184 234 220 205 199 192 36 235 182 232 225 202 191 185 43 231 186 219 229 198 187 194 50 251 216 215 259 233 236 204 57 235 190 186 225 200 201 n.d. 64 240 190 190 236 218 236 n.d. 71 233 199 178 239 245 228 n.d. 78 234 189 177 234 250 221 n.d. 85 246 196 187 243 240 224 n.d. n.d. = no data

Bleeding Assay

On different days during the treatment period, bleeding assay was performed using a Surgicutt Jr. device (ITC, New Jersey). Monkeys were placed in monkey chair with their arm placed in a steady support. The arm was lightly shaved and a sphygmomanometer was placed on the upper arm. The cuff of the sphygmomanometer was inflated to 40 mm Hg and this pressure was maintained throughout the procedure. The area on the upper arm to be incised was cleansed with an antiseptic swab and the Surgicutt Jr device was used to make an incision over the lateral aspect, volar surface of the forearm, parallel to and 5 cm below the antecubital crease. At the exact moment the incision was made, a stopwatch was started. Every 30 seconds, blood from the incision was blotted out using a blotting paper without directly touching the incision, so that formation of the platelet plug was not disturbed. Blood was blotted out every 30 seconds until blood no longer stained the paper. The stopwatch was then stopped and the bleeding time determined. The sphygmomanometer was removed from the animal's arm, the incision site was antiseptically swabbed and a wound closure strip applied. The results are provided in Table X, expressed in seconds. The results are provided in Table 120. The ISIS oligonucleotide, ISIS 416850, given with the chronic dose regimen is distinguished from the other oligonucleotides with an asterisk (*).

These data suggest that the hemorrhagic potential of the compounds provided herein is low.

TABLE 120 Bleeding assay in cynomolgus monkeys Days before/after ISIS ISIS ISIS ISIS ISIS treatment PBS ISIS 416838 416850 416858 416864 417002 416850* −14 147 200 172 154 166 185 177 −5 153 150 127 149 111 175 93 15 111 167 165 146 153 174 149 22 113 165 151 100 133 194 143 36 174 166 137 206 205 186 221 43 157 120 216 111 146 120 156 57 147 238 195 138 216 206 n.d. 64 113 131 201 113 218 146 n.d. 78 114 145 203 186 170 163 n.d. 85 147 201 201 191 203 182 n.d.

Platelet Aggregation Assay

Platelet aggregation was initiated by adding 1 mmol/L ADP and/or 3 μg collagen (depending on the collection day, as outlined in Table 121) to plasma samples, and was allowed to proceed for 10 minutes. Aggregation was characterized by recording the change in the electrical resistance or impedance and the change in the initial slope of aggregation after platelet shape change. The aggregation test was performed twice per sample on each collection day and the average value was taken. The ISIS oligonucleotide, ISIS 416850, given with the chronic dose regimen is distinguished from the other oligonucleotides with an asterisk (*).

TABLE 121 Effect of antisense oligonucleotide treatment on platelet aggregation in cynomolgus monkeys in Ohms day −5 day 15 day 36 day 43 day 57 day 64 day 78 day 85 day 85 (with (with (with (with (with (with (with (with (with collagen) ADP) ADP) collagen) ADP) collagen) ADP) ADP) collagen) PBS 17 15 7 14 16 13 12 16 17 ISIS 15 15 8 16 7 13 11 15 24 416838 ISIS 23 12 16 16 18 17 9 22 26 416850 ISIS 22 19 17 16 11 14 8 18 23 416858 ISIS 27 20 17.8 20 18 17 13 22 28 416864 ISIS 21 16 13.9 19 18 18 18 22 24 417002 ISIS 21 14 11.6 21 n.d. n.d. n.d. n.d. n.d. 416850* n.d. = no data

Body and Organ Weights

Body weights were taken once weekly throughout the dosing regimen. The measurements of each group are given in Table 122 expressed in grams. The results indicate that treatment with the antisense oligonucleotides did not cause any adverse changes in the health of the animals, which may have resulted in a significant alteration in weight compared to the PBS control. Organ weights were taken after the animals were euthanized and livers, kidneys and spleens were harvested and weighed. The results are presented in Table 123 and also show no significant alteration in weights compared to the PBS control, except for ISIS 416858, which shows increase in spleen weight. The ISIS oligonucleotide, ISIS 416850, given with the chronic dose regimen is distinguished from the other oligonucleotides with an asterisk (*).

TABLE 122 Weekly measurements of body weights (g) of cynomolgus monkeys ISIS ISIS ISIS ISIS ISIS ISIS day PBS 416838 416850 416858 416864 417002 416850* 1 2780 2720 2572 2912 2890 2640 2665 8 2615 2592 2430 2740 2784 2523 2579 15 2678 2642 2474 2760 2817 2571 2607 22 2715 2702 2514 2800 2857 2617 2661 29 2717 2689 2515 2763 2863 2622 2667 36 2738 2708 2545 2584 3327 2631 2656 43 2742 2700 2544 2607 3355 2630 2670 50 2764 2731 2613 2646 3408 2652 2679 57 2763 2737 2629 2617 3387 2654 n.d. 64 2781 2746 2642 2618 3384 2598 n.d. 71 2945 2869 2769 2865 2942 2727 n.d. 78 2815 2766 2660 2713 2822 2570 n.d. n.d. = no data

TABLE 123 Organ weights (g) of cynomolgus monkeys after antisense oligonucleotide treatment Liver Spleen Kidney PBS 46 4 11 ISIS 416838 63 5 12 ISIS 416580 64 4 16 ISIS 416858 60 12 13 ISIS 416864 53 5 14 ISIS 417002 51 5 15

Liver Function

To evaluate the impact of ISIS oligonucleotides on hepatic function, plasma concentrations of transaminases were measured using an automated clinical chemistry analyzer (Hitachi Olympus AU400e, Melville, N.Y.). Plasma concentrations of ALT (alanine transaminase) and AST (aspartate transaminase) were measured and the results are presented in Tables 124 and 125 expressed in IU/L. Those antisense oligonucleotides which did not affect an increase in ALT/AST levels above seven-fold of control levels were selected for further studies. Plasma levels of bilirubin were also measured and results are presented in Table 126 expressed in mg/dL. Those antisense oligonucleotides which did not affect an increase in levels of bilirubin more than two-fold of the control levels by antisense oligonucleotide treatment were selected for further studies. The ISIS oligonucleotide, ISIS 416850, given with the chronic dose regimen is distinguished from the other oligonucleotides with an asterisk (*).

TABLE 124 Effect of antisense oligonucleotide treatment on ALT (IU/L) in the liver of cynomolgus monkeys Days before/after ISIS ISIS ISIS ISIS ISIS treatment PBS ISIS 416838 416850 416858 416864 417002 416850* −14 57 76 54 47 54 61 80 22 39 36 41 28 37 36 42 43 36 35 43 36 36 35 41 64 38 40 60 47 43 42 n.d. 85 34 41 75 50 43 116 n.d. n.d. = no data

TABLE 125 Effect of antisense oligonucleotide treatment on AST (IU/L) in the liver of cynomolgus monkeys Days before/after ISIS ISIS ISIS ISIS ISIS treatment PBS ISIS 416838 416850 416858 416864 417002 416850* −14 71 139 81 58 76 114 100 22 43 39 45 38 41 44 39 43 38 32 50 39 40 42 40 64 35 33 56 50 46 37 n.d. 85 41 30 82. 49 56 50 n.d. n.d. = no data

TABLE 126 Effect of antisense oligonucleotide treatment on bilirubin (mg/dL) in the liver of cynomolgus monkeys Days before/after ISIS ISIS ISIS ISIS ISIS treatment PBS ISIS 416838 416850 416858 416864 417002 416850* −14 0.24 0.26 0.21 0.27 0.31 0.26 0.28 22 0.16 0.17 0.13 0.18 0.22 0.20 0.19 43 0.17 0.17 0.13 0.14 0.17 0.21 0.18 64 0.19 0.15 0.14 0.12 0.16 0.14 n.d. 85 0.20 0.13 0.14 0.14 0.17 0.12 n.d. n.d. = no data

Kidney Function

To evaluate the impact of ISIS oligonucleotides on kidney function, urine samples were collected. The ratio of urine protein to creatinine in urine samples after antisense oligonucleotide treatment was calculated and is presented in Table 127. Those antisense oligonucleotides which did not affect more than a five-fold increase in urine protein/creatinine ratios compared to the PBS control were selected for further studies.

TABLE 127 Effect of antisense oligonucleotide treatment on urine protein to creatinine ratio in cynomolgus monkeys Day 80 Day 84 PBS 0.09 0.10 ISIS 416838 0.13 0.13 ISIS 416850 0.09 0.12 ISIS 416858 0.10 0.07 ISIS 416864 0.36 0.34 ISIS 417002 0.18 0.24

Measurement of Oligonucleotide Concentration

The concentration of the full-length oligonucleotide as well as the elapsed time oligonucleotide degradation and elimination from the liver and kidney were evaluated. The method used is a modification of previously published methods (Leeds et al., 1996; Geary et al., 1999) which consist of a phenol-chloroform (liquid-liquid) extraction followed by a solid phase extraction. An internal standard (ISIS 355868, a 27-mer 2′-O-methoxyethyl modified phosphorothioate oligonucleotide, GCGTTTGCTCTTCTTCTTGCGTTTTTT, designated herein as SEQ ID NO: 270) was added prior to extraction. Tissue sample concentrations were calculated using calibration curves, with a lower limit of quantitation (LLOQ) of approximately 1.14 μg/g. Half-lives were then calculated using WinNonlin software (PHARSIGHT). The results are presented in Tables 128 and 129, expressed as μg/g liver or kidney tissue.

TABLE 128 Full-length oligonucleotide concentration (μg/g) in the liver and kidney of cynomolgus monkeys ISIS No. Kidney Liver 416838 1339 1087 416850 2845 1225 416858 1772 1061 416864 2093 1275 417002 2162 1248

TABLE 129 Total oligonucleotide concentration (μg/g) in the liver and kidney of cynomolgus monkeys ISIS No. Kidney Liver 416838 1980 1544 416850 3988 1558 416858 2483 1504 416864 3522 1967 417002 3462 1757

Hematology Assays

Blood obtained from all monkey groups were sent to Korea Institute of Toxicology (KIT) for HCT, MCV, MCH, and MCHC analysis, as well as measurements of the various blood cells, such as WBC (neutrophils, lymphocytes, monocytes, eosinophils, basophils, reticulocytes), RBC, platelets and total hemoglobin content. The results are presented in Tables 130-143. Those antisense oligonucleotides which did not affect a decrease in platelet count of more than 50% and an increase in monocyte count of more than three-fold were selected for further studies. The ISIS oligonucleotide, ISIS 416850, given with the chronic dose regimen is distinguished from the other oligonucleotides with an asterisk (*).

TABLE 130 Effect of antisense oligonucleotide treatment on WBC count (×10³/μL) in cynomolgus monkeys ISIS ISIS ISIS PBS ISIS 416838 ISIS 416850 ISIS 416858 416864 417002 416850* day −14 14 12 13 14 13 13 15 day −5 13 12 13 14 13 14 15 day 8 10 10 10 12 11 10 13 day 15 10 10 9 11 10 10 16 day 22 12 11 10 11 10 10 15 day 29 11 11 11 12 10 10 14 day 36 10 10 10 12 10 11 16 day 43 10 10 9 11 10 10 15 day 50 12 11 11 13 12 13 15 day 57 11 12 11 13 12 12 n.d. day 64 11 13 11 12 11 11 n.d. day 71 15 15 15 13 14 12 n.d. day 78 10 11 12 11 11 9 n.d. day 85 10 12 15 11 12 10 n.d. n.d. = no data

TABLE 131 Effect of antisense oligonucleotide treatment on RBC count (×10⁶/μL) in cynomolgus monkeys ISIS ISIS ISIS PBS ISIS 416838 ISIS 416850 ISIS 416858 416864 417002 416850* day −14 5.7 5.6 5.3 5.6 5.5 5.6 5.5 day −5 5.7 5.6 5.5 5.6 5.6 5.6 5.5 day 8 5.7 5.7 5.4 5.6 5.7 5.6 5.5 day 15 5.6 5.6 5.3 5.4 5.7 5.4 5.3 day 22 5.5 5.4 5 5.3 5.3 5.2 5.1 day 29 5.6 5.3 4.9 5.3 5.3 5.2 5.2 day 36 5.7 5.5 5.3 5.5 5.6 5.4 5.3 day 43 5.7 5.6 5.2 5.5 5.5 5.4 5.2 day 50 5.8 5.5 5.2 5.5 5.6 5.4 5.3 day 57 5.7 5.5 5.2 5.6 5.5 4.9 n.d. day 64 5.8 5.6 5.4 5.7 5.6 5.4 n.d. day 71 5.6 5.5 5.4 5.6 5.6 5.5 n.d. day 78 5.6 5.4 5.3 5.4 5.3 5.4 n.d. day 85 5.6 5.5 5.5 5.5 5.4 5.4 n.d. n.d. = no data

TABLE 132 Effect of antisense oligonucleotide treatment on hemoglobin (g/dL) in cynomolgus monkeys ISIS ISIS ISIS ISIS ISIS ISIS PBS 416838 416850 416858 416864 417002 416850* day −14 13.2 12.9 12.4 13.2 12.7 13.0 12.8 day −5 13.1 13.1 12.7 13.2 13.0 13.2 12.8 day 8 13.1 12.9 12.4 12.8 12.7 12.8 12.5 day 15 12.9 12.9 12.1 12.6 12.8 12.3 12.2 day 22 12.7 12.5 11.6 12.4 12.1 12.1 11.7 day 29 12.8 12.4 11.5 12.3 12.1 12.0 12.0 day 36 13.0 12.8 12.2 12.6 12.5 12.5 12.3 day 43 12.9 12.7 11.8 12.4 12.2 12.3 11.8 day 50 12.6 12.3 11.8 12.2 12.1 12.3 11.9 day 57 13.1 12.6 12.1 12.7 12.3 11.3 n.d. day 64 13.1 12.6 12.3 12.8 12.1 12.2 n.d. day 71 12.9 12.7 12.3 12.7 12.2 12.5 n.d. day 78 13.0 12.5 12.2 12.4 11.9 12.4 n.d. day 85 13.2 12.4 12.7 11.9 12.3 12.2 n.d. n.d. = no data

TABLE 133 Effect of antisense oligonucleotide treatment on hematocrit (%) in cynomolgus monkeys ISIS ISIS ISIS ISIS ISIS ISIS PBS 416838 416850 416858 416864 417002 416850* day −14 46 42 41 43 43 44 44 day −5 44 42 43 42 44 45 43 day 8 44 43 43 43 44 44 43 day 15 44 42 40 40 42 40 40 day 22 45 43 41 41 42 41 40 day 29 46 43 41 41 43 42 42 day 36 46 43 42 40 42 42 41 day 43 46 43 40 40 42 41 40 day 50 48 44 42 41 44 43 42 day 57 46 43 42 41 42 38 n.d. day 64 47 44 43 42 42 41 n.d. day 71 46 44 43 42 44 43 n.d. day 78 43 41 41 39 39 40 n.d. day 85 43 42 42 39 40 41 n.d. n.d. = no data

TABLE 134 Effect of antisense oligonucleotide treatment on MCV (fL) in cynomolgus monkeys ISIS ISIS ISIS ISIS ISIS ISIS PBS 416838 416850 416858 416864 417002 416850* day −14 81 77 78 77 79 79 81 day −5 78 76 77 75 79 80 78 day 8 77 77 80 77 78 79 79 day 15 78 75 76 74 74 76 75 day 22 84 80 83 77 79 79 79 day 29 83 81 83 78 80 81 82 day 36 81 78 80 75 76 78 76 day 43 80 78 79 74 77 77 77 day 50 84 80 83 76 79 80 80 day 57 82 79 80 74 77 80 n.d. day 64 81 79 79 73 75 76 n.d. day 71 84 80 80 75 79 78 n.d. day 78 78 76 79 72 74 75 n.d. day 85 77 77 77 72 74 76 n.d. n.d. = no data

TABLE 135 Effect of antisense oligonucleotide treatment on MCH (pg) in cynomolgus monkeys ISIS ISIS ISIS ISIS ISIS ISIS PBS 416838 416850 416858 416864 417002 416850* day −14 23 23 23 24 23 24 24 day −5 23 23 23 23 23 24 23 day 8 23 23 23 23 23 23 23 day 15 23 23 23 23 23 23 23 day 22 23 23 24 24 23 23 23 day 29 23 23 23 23 23 23 23 day 36 23 23 23 23 23 23 23 day 43 23 23 23 23 22 23 23 day 50 22 23 23 23 22 23 23 day 57 23 23 23 22 23 23 n.d. Day 64 23 23 22 22 23 22 n.d. Day 71 23 23 23 22 23 23 n.d. Day 78 23 23 23 23 23 23 n.d. Day 85 23 23 22 22 23 23 n.d. n.d. = no data

TABLE 136 Effect of antisense oligonucleotide treatment on MCHC (g/dL) in cynomolgus monkeys ISIS ISIS ISIS ISIS ISIS ISIS PBS 416838 416850 416858 416864 417002 416850* day −14 29 30 30 31 29 30 29 day −5 30 31 30 31 29 30 30 day 8 30 30 29 30 29 29 29 day 15 30 31 30 31 30 31 30 day 22 28 29 28 30 29 29 29 day 29 28 29 28 30 29 29 28 day 36 28 30 29 31 30 30 30 day 43 28 30 29 31 29 30 30 day 50 26 28 28 30 28 29 29 day 57 29 29 29 31 29 29 n.d. day 64 28 29 29 30 29 30 n.d. day 71 28 29 28 30 28 29 n.d. day 78 30 30 29 32 30 31 n.d. day 85 31 30 30 31 30 30 n.d. n.d. = no data

TABLE 137 Effect of antisense oligonucleotide treatment on platelet count (×10³/μL) in cynomolgus monkeys ISIS ISIS ISIS ISIS ISIS ISIS PBS 416838 416850 416858 416864 417002 416850* day −14 349 377 528 419 434 442 387 day −5 405 425 573 463 456 466 434 day 8 365 387 548 391 438 435 401 day 15 375 387 559 400 439 410 396 day 22 294 319 466 316 364 377 347 day 29 311 337 475 336 397 410 370 day 36 326 370 505 371 428 415 379 day 43 336 365 490 342 351 393 391 day 50 379 372 487 331 419 389 351 day 57 345 371 528 333 409 403 n.d. day 64 329 358 496 295 383 436 n.d. day 71 322 365 465 286 394 490 n.d. day 78 309 348 449 262 366 432 n.d. day 85 356 344 458 267 387 418 n.d. n.d. = no data

TABLE 138 Effect of antisense oligonucleotide treatment on reticulocytes (%) in cynomolgus monkeys ISIS ISIS ISIS ISIS ISIS ISIS PBS 416838 416850 416858 416864 417002 416850* day −14 1.4 1.0 1.7 1.0 0.9 0.9 1.1 day −5 1.0 0.9 1.2 0.9 0.9 0.8 0.8 day 8 1.0 1.2 1.2 1.2 0.8 1.1 1.1 day 15 1.5 1.2 1.9 1.6 0.8 1.1 1.0 day 22 1.2 1.2 1.9 1.3 0.9 1.2 1.0 day 29 1.6 1.6 2.5 1.5 1.3 1.6 1.4 day 36 1.7 1.6 2.2 1.6 1.3 1.3 1.3 day 43 1.3 1.2 1.6 1.3 1.1 1.1 1.0 day 50 1.6 1.6 2.7 1.5 1.3 1.6 1.2 day 57 1.8 1.5 2.0 1.4 1.0 4.6 n.d. day 64 1.3 1.3 1.7 1.0 0.8 1.3 n.d. day 71 1.6 1.3 1.8 1.3 1.0 1.3 n.d. day 78 1.5 1.4 1.8 1.2 1.2 1.3 n.d. day 85 1.5 1.5 2.3 1.3 1.5 1.4 n.d. n.d. = no data

TABLE 139 Effect of antisense oligonucleotide treatment on neutrophils (%) in cynomolgus monkeys ISIS ISIS ISIS ISIS ISIS ISIS PBS 416838 416850 416858 416864 417002 416850* day −14 40 36 49 37 53 43 48 day −5 37 35 52 46 51 43 53 day 8 54 42 57 51 52 46 53 day 15 49 43 58 54 59 57 73 day 22 41 37 57 47 59 55 64 day 29 44 36 53 43 44 45 42 day 36 37 39 57 47 58 61 72 day 43 40 30 50 45 57 57 61 day 50 36 31 45 46 49 61 62 day 57 41 32 49 44 57 54 n.d. day 64 40 30 41 37 49 55 n.d. day 71 38 28 27 26 42 34 n.d. day 78 42 35 42 39 48 51 n.d. day 85 30 22 60 40 39 36 n.d. n.d. = no data

TABLE 140 Effect of antisense oligonucleotide treatment on lymphocytes (%) in cynomolgus monkeys ISIS ISIS ISIS ISIS ISIS ISIS PBS 416838 416850 416858 416864 417002 416850* day −14 54 59 47 58 42 53 47 day −5 56 59 43 49 44 53 43 day 8 43 54 39 45 45 50 44 day 15 47 53 38 43 38 40 24 day 22 54 59 39 49 37 41 33 day 29 51 59 43 51 51 50 53 day 36 58 57 39 49 38 35 26 day 43 55 65 45 51 39 39 36 day 50 59 64 49 48 46 34 35 day 57 55 63 45 51 39 40 n.d. day 64 56 64 53 56 46 39 n.d. day 71 56 65 61 66 52 59 n.d. day 78 53 60 51 54 46 41 n.d. day 85 63 72 34 52 54 56 n.d. n.d. = no data

TABLE 141 Effect of antisense oligonucleotide treatment on eosinophils (%) in cynomolgus monkeys ISIS ISIS ISIS ISIS ISIS ISIS PBS 416838 416850 416858 416864 417002 416850* day −14 1.3 0.6 1.0 0.7 1.0 0.3 0.5 day −5 1.5 0.6 1.6 1.3 0.9 0.3 0.7 day 8 0.9 0.4 1.1 0.3 0.7 0.2 0.5 day 15 0.7 0.3 1.0 0.3 0.5 0.1 0.2 day 22 0.9 0.5 0.7 0.6 0.9 0.3 0.5 day 29 0.9 0.3 1.2 0.6 0.9 0.3 0.8 day 36 0.9 0.5 1.7 0.4 0.6 0.2 0.4 day 43 0.9 0.6 1.2 0.3 0.6 0.2 0.4 day 50 1.2 0.8 1.2 0.4 0.7 0.1 0.3 day 57 0.7 0.6 1.0 0.3 0.4 0.2 n.d. day 64 1.0 0.7 1.3 0.4 0.7 0.2 n.d. day 71 1.6 0.8 1.8 0.9 1.1 0.3 n.d. day 78 1.0 0.9 1.0 0.5 1.2 0.1 n.d. day 85 1.3 1.5 1.2 0.6 1.6 0.2 n.d. n.d. = no data

TABLE 142 Effect of antisense oligonucleotide treatment on monocytes (%) in cynomolgus monkeys ISIS ISIS ISIS ISIS ISIS ISIS PBS 416838 416850 416858 416864 417002 416850* day −14 3.3 3.1 2.3 2.8 2.8 3.0 2.9 day −5 3.8 3.6 2.8 2.8 3.3 3.2 2.4 day 8 2.3 2.5 1.8 2.7 2.1 3.3 1.8 day 15 2.7 2.4 2.0 2.2 2.4 2.3 1.5 day 22 3.4 2.9 2.4 2.8 2.8 3.1 1.9 day 29 3.3 3.2 2.7 3.8 3.4 3.5 2.7 day 36 3.1 2.5 2.1 2.9 2.3 2.6 1.5 day 43 3.5 3.3 2.6 3.1 2.1 2.8 1.8 day 50 2.6 3.2 3.7 4.6 2.9 3.1 1.8 day 57 2.6 3.2 n.d. 3.2 3.8 2.4 3.6 n.d. day 64 2.6 3.5 n.d. 3.5 4.4 2.8 4.0 n.d. day 71 3.4 4.3 n.d. 4.7 4.9 3.7 4.7 n.d. day 78 3.3 3.6 n.d. 4.5 4.9 3.7 4.7 n.d. day 85 4.4 3.7 n.d. 3.5 6.1 3.7 5.3 n.d. n.d. = no data

TABLE 143 Effect of antisense oligonucleotide treatment on basophils (%) in cynomolgus monkeys ISIS ISIS ISIS ISIS ISIS ISIS PBS 416838 416850 416858 416864 417002 416850* day −14 0.3 0.2 0.2 0.3 0.2 0.3 0.2 day −5 0.3 0.3 0.2 0.3 0.2 0.3 0.3 day 8 0.2 0.2 0.2 0.3 0.2 0.3 0.3 day 15 0.3 0.3 0.2 0.2 0.2 0.2 0.2 day 22 0.2 0.2 0.2 0.2 0.2 0.2 0.1 day 29 0.3 0.2 0.2 0.2 0.3 0.2 0.3 day 36 0.3 0.4 0.3 0.3 0.3 0.2 0.1 day 43 0.3 0.4 0.3 0.3 0.4 0.3 0.2 day 50 0.4 0.3 0.3 0.4 0.4 0.3 0.2 day 57 0.2 0.3 0.4 0.2 0.3 0.3 n.d. day 64 0.3 0.4 0.4 0.4 0.4 0.2 n.d. day 71 0.2 0.5 0.3 0.4 0.4 0.3 n.d. day 78 0.2 0.4 0.3 0.4 0.3 0.3 n.d. day 85 0.3 0.3 0.3 0.3 0.4 0.3 n.d. n.d. = no data

Cytokine and Chemokine Assays

Blood samples obtained from the monkey groups treated with PBS, ISIS 416850 and ISIS 416858 administered in the escalating dose regimen were sent to Pierce Biotechnology (Woburn, Mass.) for measurement of chemokine and cytokine levels. Levels of IL-1β, IL-6, IFN-γ, and TNF-α were measured using the respective primate antibodies and levels of IL-8, MIP-1α, MCP-1, MIP-1β and RANTES were measured using the respective cross-reacting human antibodies. Measurements were taken 14 days before the start of treatment and on day 85, when the monkeys were euthanized. The results are presented in Tables 144 and 145.

TABLE 144 Effect of antisense oligonucleotide treatment on cytokine/chemokine levels (pg/mL) in cynomolgus monkeys on day −14 IL-1β IL-6 IFN-γ TNF-α IL-8 MIP-1α MCP-1 MIP-1β RANTES PBS 16 10 114 7 816 54 1015 118 72423 ISIS 416850 3 30 126 14 1659 28 1384 137 75335 ISIS 416858 5 9 60 9 1552 36 1252 122 112253

TABLE 145 Effect of antisense oligonucleotide treatment on cytokine/chemokine levels (pg/mL) in cynomolgus monkeys on day 85 IL-1β IL-6 IFN-γ TNF-α IL-8 MIP-1α MCP-1 MIP-1β RANTES PBS 7 4 102 34 87 23 442 74 84430 ISIS 416850 13 17 18 27 172 41 2330 216 83981 ISIS 416858 5 25 18 45 303 41 1752 221 125511

Example 46 Pharmacologic Effect of Antisense Oligonucleotides Targeting Human Factor 11 in Cynomolgus Monkeys

Several antisense oligonucleotides chosen from the rodent tolerability studies (Examples 41-44) were tested in cynomolgus monkeys to determine their pharmacologic effects, relative efficacy on Factor 11 activity and tolerability in a cynomolgus monkey model. The antisense oligonucleotides were also compared to ISIS 416850 and ISIS 416858 selected from the monkey study described earlier (Example 45). All the ISIS oligonucleotides used in this study target human Factor 11 mRNA and are also fully cross-reactive with the rhesus monkey gene sequence (see Tables 44 and 46). It is expected that the rhesus monkey ISIS oligonucleotides are fully cross-reactive with the cynomolgus monkey gene sequence as well. At the time the study was undertaken, the cynomolgus monkey genomic sequence was not available in the National Center for Biotechnology Information (NCBI) database; therefore, cross-reactivity with the cynomolgus monkey gene sequence could not be confirmed.

Treatment

Groups, each consisting of two male and two female monkeys, were injected subcutaneously with 25 mg/kg of ISIS 416850, ISIS 449709, ISIS 445522, ISIS 449710, ISIS 449707, ISIS 449711, ISIS 449708, 416858, and ISIS 445531. Antisense oligonucleotide was administered to the monkeys at 25 mg/kg three times per week for week 1 and 25 mg/kg twice per week for weeks 2 to 8. A control group, consisting of two male and two female monkeys was injected subcutaneously with PBS according to the same dosing regimen. Body weights were taken 14 days and 7 days before the start of treatment and were then measured weekly throughout the treatment period. Blood samples were collected 14 days and 5 days before the start of treatment and subsequently several times during the dosing regimen for PT and aPTT measurements, and measurement of various hematologic factors. On day 55, the monkeys were euthanized by exsanguination while under deep anesthesia, and organs harvested for further analysis.

RNA Analysis

On day 55, RNA was extracted from liver tissue for real-time PCR analysis of Factor 11 using primer probe set LTS00301. Results are presented as percent inhibition of Factor 11, relative to PBS control. As shown in Table 146, treatment with ISIS 416850, ISIS 449709, ISIS 445522, ISIS 449710, ISIS 449707, ISIS 449708, ISIS 416858, and ISIS 445531 resulted in significant reduction of Factor 11 mRNA in comparison to the PBS control.

TABLE 146 Inhibition of Factor 11 mRNA in the cynomolgus monkey liver relative to the PBS control Oligo ID % inhibition 416850 68 449709 69 445522 89 449710 52 449707 47 449711 0 449708 46 416858 89 445531 66

Protein Analysis

Plasma samples from all monkey groups taken on different days were analyzed by a sandwich-style ELISA assay (Affinity Biologicals Inc.) using an affinity-purified polyclonal anti-Factor 11 antibody as the capture antibody and a peroxidase-conjugated polyclonal anti-Factor 11 antibody as the detecting antibody. Monkey plasma was diluted 1:50 for the assay. Peroxidase activity was expressed by incubation with the substrate o-phenylenediamine. The color produced was quantified using a microplate reader at 490 nm and was considered to be proportional to the concentration of Factor 11 in the samples.

The results are presented in Table 147, expressed as percentage reduction relative to that of the PBS control. Treatment with ISIS 416850, ISIS 449709, ISIS 445522, and ISIS 416858 resulted in a time-dependent decrease in protein levels.

TABLE 147 Inhibition of Factor 11 protein in the cynomolgus monkey liver relative to the PBS control ISIS No. Day −14 Day −5 Day 10 Day 17 Day 24 Day 31 Day 38 Day 45 Day 52 Day 55 416850 0 0 20 31 38 52 51 53 53 58 449709 1 0 27 35 44 45 46 48 47 50 445522 2 0 36 50 61 70 73 77 80 82 449710 1 0 10 14 17 25 20 23 4 24 449707 0 0 16 19 21 29 28 35 29 32 449711 0 1 5 3 6 9 2 4 3 5 449708 1 0 7 15 3 14 9 2 6 6 416858 4 0 36 49 62 68 74 79 81 81 445531 0 1 9 22 23 27 29 32 32 37 PT and aPTT Assay

PT and aPTT were measured using platelet poor plasma (PPP) from mice treated with ISIS oligonucleotides. PT and aPTT values are provided in Tables 148 and 149 and are reported as International Normalized Ratio (INR) values. INR values for PT and aPTT were determined by dividing the PT or aPTT value for each experimental group by the PT or aPTT for the PBS treated group. This ratio was then raised to the power of the International Sensitivity Index (ISI) of the tissue factor used. As shown in Table 148, PT was not significantly prolonged in mice treated with ISIS oligonucleotides. However, aPTT was significantly prolonged in groups treated with ISIS 416850, ISIS 445522, and ISIS 416858, as presented in Table 149. These data suggest that antisense reduction of Factor 11 affects the contact activation pathway, but not the extrinsic pathway of blood coagulation. Therefore, antisense reduction of Factor 11 with these ISIS oligonucleotides is useful for inhibiting the formation of a thrombus or clot in response to an abnormal vessel wall, but not in response to tissue injury.

TABLE 148 Effect of antisense oligonucleotide treatment on PT ratio in cynomolgus monkeys Day −14 Day −5 Day 10 Day 17 Day 24 Day 31 Day 38 Day 45 Day 52 Day 55 ISIS 416850 1.02 1.00 0.99 1.00 0.97 1.00 1.01 1.00 1.02 1.07 ISIS 449709 1.00 0.96 0.95 0.95 0.95 0.95 0.97 0.97 0.99 1.03 ISIS 445522 1.00 0.94 0.95 0.96 0.94 0.96 0.97 0.96 0.98 1.01 ISIS 449710 1.03 0.96 0.98 1.00 0.97 0.98 0.99 0.97 0.98 1.06 ISIS 449707 1.01 0.94 0.95 0.97 0.95 0.96 1.00 0.96 0.96 1.00 ISIS 449711 1.00 0.95 0.94 0.95 0.94 0.98 1.02 1.01 1.00 1.07 ISIS 449708 1.03 0.95 0.98 1.00 0.95 1.06 0.99 0.99 0.99 1.04 ISIS 416858 1.01 0.96 0.96 0.98 0.95 1.00 0.97 1.00 0.99 1.01 ISIS 445531 1.06 1.00 1.00 1.06 1.02 1.04 1.03 1.01 1.04 1.06

TABLE 149 Effect of antisense oligonucleotide treatment on aPTT ratio in cynomolgus monkeys Day −14 Day −5 Day 10 Day 17 Day 24 Day 31 Day 38 Day 45 Day 52 Day 55 ISIS 416850 0.99 0.90 0.98 1.01 1.05 1.22 1.25 1.34 1.32 1.45 ISIS 449709 0.99 0.91 0.99 1.03 1.05 1.08 1.08 1.15 1.09 1.17 ISIS 445522 0.96 0.91 1.06 1.10 1.14 1.25 1.32 1.39 1.39 1.42 ISIS 449710 1.07 0.98 1.00 0.97 1.00 1.04 1.02 1.06 1.03 1.07 ISIS 449707 0.90 0.87 0.92 0.94 0.93 0.95 0.99 1.00 0.99 1.04 ISIS 449711 0.94 0.96 0.92 0.90 0.92 0.89 0.93 0.94 0.92 0.96 ISIS 449708 1.07 1.01 1.06 1.05 1.01 1.09 1.06 1.06 1.08 1.11 ISIS 416858 1.03 0.96 1.07 1.13 1.21 1.32 1.41 1.49 1.53 1.61 ISIS 445531 1.00 0.89 0.95 1.05 1.00 1.07 1.06 1.13 1.15 1.19

Body and Organ Weights

Body weights of each group are given in Table 150 expressed in grams. The results indicate that treatment with the antisense oligonucleotides did not cause any adverse changes in the health of the animals, which may have resulted in a significant alteration in weight compared to the PBS control. Organ weights were taken after the animals were euthanized on day 55, and livers, kidneys and spleens were harvested. The results are presented in Table 150 expressed as a percentage of the body weight and also show no significant alteration in weights compared to the PBS control, with the exception of ISIS 449711, which caused increase in spleen weight.

TABLE 150 Weekly measurements of body weights (g) of cynomolgus monkeys ISIS ISIS ISIS ISIS ISIS ISIS ISIS ISIS ISIS Days PBS 416850 449709 445522 449710 449707 449711 449708 416858 445531 −14 2069 2061 2044 2050 2097 2072 2049 2096 2073 2079 −7 2107 2074 2093 2042 2114 2083 2105 2163 2092 2092 1 2131 2083 2112 2047 2131 2107 2123 2130 2115 2125 8 2186 2072 2075 2094 2120 2088 2123 2148 2149 2119 15 2201 2147 2085 2092 2145 2120 2103 2125 2162 2109 22 2206 2139 2117 2114 2177 2142 2171 2110 2188 2143 29 2204 2159 2068 2125 2149 2155 2203 2095 2196 2148 36 2246 2136 2064 2121 2180 2158 2227 2100 2210 2191 43 2304 2186 2106 2142 2227 2197 2251 2125 2238 2233 50 2274 2143 2147 2127 2201 2185 2227 2076 2225 2197

TABLE 151 Organ weights (g) of cynomolgus monkeys after antisense oligonucleotide treatment Liver Spleen Kidney PBS 2.3 0.16 0.48 ISIS 416850 2.5 0.17 0.51 ISIS 449709 2.6 0.21 0.57 ISIS 445522 2.6 0.23 0.55 ISIS 449710 2.6 0.24 0.58 ISIS 449707 2.5 0.24 0.53 ISIS 449711 2.6 0.32 0.54 ISIS 449708 2.6 0.19 0.60 ISIS 416858 2.6 0.24 0.47 ISIS 445531 2.8 0.24 0.49

Liver Function

To evaluate the impact of ISIS oligonucleotides on hepatic function, plasma concentrations of ALT and AST were measured using an automated clinical chemistry analyzer (Hitachi Olympus AU400e, Melville, N.Y.). Plasma concentrations of alanine transaminase (ALT) and aspartate transaminase (AST) were measured and the results are presented in Tables 152 and 153 expressed in IU/L. Plasma levels of bilirubin were also measured and results are presented in Table 154 expressed in mg/dL. As observed in Tables 152-154, there were no significant increases in any of the liver metabolic markers after antisense oligonucleotide treatment.

TABLE 152 Effect of antisense oligonucleotide treatment on ALT (IU/L) in the liver of cynomolgus monkeys Day −14 Day −5 Day 31 Day 55 PBS 57 55 53 57 ISIS 416850 48 42 45 55 ISIS 449709 73 77 65 102 ISIS 445522 43 45 40 60 ISIS 449710 37 42 37 45 ISIS 449707 54 56 52 63 ISIS 449711 49 137 48 54 ISIS 449708 48 54 44 46 ISIS 416858 43 66 46 58 ISIS 445531 84 73 57 73

TABLE 153 Effect of antisense oligonucleotide treatment on AST (IU/L) in the liver of cynomolgus monkeys Day −14 Day −5 Day 31 Day 55 PBS 65 45 44 47 ISIS 416850 62 45 46 57 ISIS 449709 62 51 45 71 ISIS 445522 62 47 46 79 ISIS 449710 52 38 37 64 ISIS 449707 64 53 50 52 ISIS 449711 58 78 47 47 ISIS 449708 74 53 56 50 ISIS 416858 64 100 60 69 ISIS 445531 78 46 47 49

TABLE 154 Effect of antisense oligonucleotide treatment on bilirubin (mg/dL) in the liver of cynomolgus monkeys Day −14 Day −5 Day 31 Day 55 PBS 0.25 0.20 0.20 0.17 ISIS 416850 0.26 0.22 0.26 0.17 ISIS 449709 0.24 0.19 0.15 0.18 ISIS 445522 0.24 0.20 0.14 0.18 ISIS 449710 0.24 0.19 0.15 0.22 ISIS 449707 0.27 0.19 0.13 0.16 ISIS 449711 0.23 0.16 0.13 0.13 ISIS 449708 0.27 0.21 0.14 0.14 ISIS 416858 0.25 0.23 0.16 0.16 ISIS 445531 0.22 0.18 0.13 0.11

Kidney Function

To evaluate the impact of ISIS oligonucleotides on kidney function, urine samples were collected on different days. BUN levels were measured at various time points using an automated clinical chemistry analyzer (Hitachi Olympus AU400e, Melville, N.Y.) and the results are presented in Table 155. The ratio of urine protein to creatinine in urine samples after antisense oligonucleotide treatment was also calculated for day 49 and results are presented in Table 156. As observed in Tables 155 and 156, there were no significant increases in any of the kidney metabolic markers after antisense oligonucleotide treatment.

TABLE 155 Effect of antisense oligonucleotide treatment on BUN levels (mg/dL) in cynomolgus monkeys Day −14 Day −5 Day 31 Day 55 PBS 22 21 22 22 ISIS 416850 24 23 21 26 ISIS 449709 22 21 20 28 ISIS 445522 23 22 22 22 ISIS 449710 19 19 19 23 ISIS 449707 25 21 21 20 ISIS 449711 26 22 20 23 ISIS 449708 25 23 23 23 ISIS 416858 25 24 23 24 ISIS 445531 22 18 20 22

TABLE 156 Effect of antisense oligonucleotide treatment on urine protein to creatinine ratio in cynomolgus monkeys Urine protein/ creatinine ratio PBS 0.02 ISIS 416850 0.08 ISIS 449709 0.05 ISIS 445522 0.01 ISIS 449710 0.00 ISIS 449707 0.03 ISIS 449711 0.01 ISIS 449708 0.00 ISIS 416858 0.05 ISIS 445531 0.08

Hematology Assays

Blood obtained from all the monkey groups on different days were sent to Korea Institute of Toxicology (KIT) for HCT, MCV, MCH, and MCHC measurements, as well as measurements of the various blood cells, such as WBC (neutrophils and monocytes), RBC and platelets, as well as total hemoglobin content. The results are presented in Tables 157-166.

TABLE 157 Effect of antisense oligonucleotide treatment on HCT (%) in cynomolgus monkeys Day −14 Day −5 Day 17 Day 31 Day 45 Day 55 PBS 40 42 43 43 41 40 ISIS 416850 41 44 42 42 42 40 ISIS 449709 41 42 43 42 41 40 ISIS 445522 42 42 41 43 41 39 ISIS 449710 41 44 43 44 43 41 ISIS 449707 40 43 42 43 43 42 ISIS 449711 41 41 42 39 39 38 ISIS 449708 41 44 44 43 44 42 ISIS 416858 41 44 43 43 41 39 ISIS 445531 41 42 43 41 41 41

TABLE 158 Effect of antisense oligonucleotide treatment on platelet count (×100/μL) in cynomolgus monkeys Day −14 Day −5 Day 17 Day 31 Day 45 Day 55 PBS 361 441 352 329 356 408 ISIS 416850 462 517 467 507 453 396 ISIS 449709 456 481 449 471 418 441 ISIS 445522 433 512 521 425 403 333 ISIS 449710 411 463 382 422 313 360 ISIS 449707 383 464 408 408 424 399 ISIS 449711 410 431 325 309 257 259 ISIS 449708 387 517 444 378 381 348 ISIS 416858 369 433 358 289 287 257 ISIS 445531 379 416 380 376 345 319

TABLE 159 Effect of antisense oligonucleotide treatment on neutrophils (%) in cynomolgus monkeys Day −14 Day −5 Day 17 Day 31 Day 45 Day 55 PBS 81 84 75 75 91 118 ISIS 416850 88 109 95 100 85 108 ISIS 449709 73 101 89 81 77 115 ISIS 445522 61 84 81 66 69 125 ISIS 449710 93 86 80 94 97 132 ISIS 449707 85 106 80 89 89 98 ISIS 449711 64 71 52 58 45 70 ISIS 449708 73 84 61 57 61 75 ISIS 416858 65 84 54 54 61 73 ISIS 445531 60 80 85 116 93 91

TABLE 160 Effect of antisense oligonucleotide treatment on monocytes (%) in cynomolgus monkeys Day −14 Day −5 Day 17 Day 31 Day 45 Day 55 PBS 1.9 2.8 3.1 2.8 3.9 2.2 ISIS 416850 1.9 2.9 3.2 3.7 3.8 3.4 ISIS 449709 4.0 2.0 3.0 2.8 3.6 3.4 ISIS 445522 2.1 2.3 3.6 3.9 4.4 3.0 ISIS 449710 1.3 2.0 2.5 2.4 3.4 1.6 ISIS 449707 1.3 2.3 3.2 4.2 4.0 4.8 ISIS 449711 1.2 2.3 5.9 6.9 7.6 7.8 ISIS 449708 1.7 2.6 5.4 5.8 7.0 6.2 ISIS 416858 2.0 2.7 4.0 4.7 4.6 4.6 ISIS 445531 1.3 2.2 3.4 4.1 4.4 4.1

TABLE 161 Effect of antisense oligonucleotide treatment on hemoglobin content (g/dL) in cynomolgus monkeys Day −14 Day −5 Day 17 Day 31 Day 45 Day 55 PBS 12.3 12.5 12.9 12.7 12.4 12.1 ISIS 416850 13.0 13.5 13.3 13.1 13.1 12.7 ISIS 449709 12.8 12.8 13.2 13.1 12.6 12.5 ISIS 445522 13.3 12.7 12.7 12.9 12.6 12.0 ISIS 449710 13.0 13.2 13.4 13.1 13.0 12.7 ISIS 449707 12.7 12.8 12.7 12.7 12.9 12.6 ISIS 449711 12.7 12.7 12.5 11.8 11.5 11.3 ISIS 449708 13.0 13.2 13.5 13.0 13.3 13.0 ISIS 416858 12.8 13.0 13.0 12.8 12.3 12.0 ISIS 445531 12.6 12.6 12.7 12.3 12.0 12.1

TABLE 162 Effect of antisense oligonucleotide treatment on WBC count (×10³/μL) in cynomolgus monkeys Day −14 Day −5 Day 17 Day 31 Day 45 Day 55 PBS 10 10 11 12 11 12 ISIS 416850 12 13 11 12 12 10 ISIS 449709 11 10 11 11 11 10 ISIS 445522 10 9 11 13 10 11 ISIS 449710 11 11 12 12 11 15 ISIS 449707 13 11 12 11 12 8 ISIS 449711 13 12 10 9 9 7 ISIS 449708 14 10 11 11 10 10 ISIS 416858 10 11 10 9 8 9 ISIS 445531 20 15 17 17 20 15

TABLE 163 Effect of antisense oligonucleotide treatment on RBC count (×10⁶/μL) in cynomolgus monkeys Day −14 Day −5 Day 17 Day 31 Day 45 Day 55 PBS 5.6 5.6 5.8 5.8 5.6 5.5 ISIS 416850 5.5 5.7 5.6 5.6 5.7 5.6 ISIS 449709 5.8 5.8 5.9 5.9 5.7 5.7 ISIS 445522 5.9 5.6 5.6 5.8 5.7 5.4 ISIS 449710 5.6 5.8 5.8 5.8 5.7 5.6 ISIS 449707 5.7 5.8 5.7 5.7 5.9 5.8 ISIS 449711 5.6 5.7 5.6 5.4 5.4 5.3 ISIS 449708 5.7 5.9 5.9 5.8 6.0 5.8 ISIS 416858 5.5 5.5 5.6 5.6 5.5 5.3 ISIS 445531 5.7 5.7 5.8 5.6 5.5 5.6

TABLE 164 Effect of antisense oligonucleotide treatment on MCV (fL) in cynomolgus monkeys Day −14 Day −5 Day 17 Day 31 Day 45 Day 55 PBS 72 74 75 73 73 73 ISIS 416850 74 77 76 75 75 73 ISIS 449709 72 74 73 73 71 71 ISIS 445522 72 74 74 75 73 72 ISIS 449710 75 77 75 75 75 73 ISIS 449707 71 75 74 74 73 73 ISIS 449711 73 74 75 73 73 73 ISIS 449708 73 75 75 75 74 74 ISIS 416858 75 79 78 76 75 75 ISIS 445531 72 74 75 75 75 74

TABLE 165 Effect of antisense oligonucleotide treatment on MCH (pg) in cynomolgus monkeys Day −14 Day −5 Day 17 Day 31 Day 45 Day 55 PBS 22.1 22.4 22.3 22.1 22.0 22.0 ISIS 416850 23.7 23.7 23.7 23.3 22.7 22.9 ISIS 449709 22.4 22.3 22.5 22.2 21.0 22.0 ISIS 445522 22.6 22.5 22.8 22.4 22.4 22.2 ISIS 449710 23.0 22.8 23.1 22.6 21.8 22.7 ISIS 449707 22.2 22.2 22.1 22.1 22.6 21.9 ISIS 449711 22.6 22.7 22.2 22.1 21.7 21.3 ISIS 449708 22.9 22.7 22.9 22.7 22.2 22.5 ISIS 416858 23.2 23.5 23.1 23.0 22.2 22.8 ISIS 445531 22.2 22.2 22.1 22.0 21.6 21.7

TABLE 166 Effect of antisense oligonucleotide treatment on MCHC (g/dL) in cynomolgus monkeys Day −14 Day −5 Day 17 Day 31 Day 45 Day 55 PBS 30.8 30.0 30.1 29.9 30.3 30.2 ISIS 416850 32.0 30.7 31.3 31.0 31.0 30.9 ISIS 449709 31.4 30.3 30.7 30.7 31.1 31.2 ISIS 445522 31.4 30.4 30.9 30.0 30.7 31.0 ISIS 449710 31.2 29.7 30.7 30.1 30.4 31.1 ISIS 449707 31.4 29.8 30.0 29.8 29.8 30.0 ISIS 449711 31.0 30.7 29.9 29.8 29.6 29.5 ISIS 449708 31.4 30.2 30.7 29.9 30.6 31.8 ISIS 416858 31.1 29.8 29.9 31.0 30.3 30.4 ISIS 445531 30.9 30.0 29.5 29.7 29.0 29.6

Cytokine and Chemokine Assays

Blood samples obtained from all monkey groups were sent to Pierce Biotechnology (Woburn, Mass.) for measurements of chemokine and cytokine levels. Levels of IL-1β, IL-6, IFN-γ, and TNF-α were measured using the respective primate antibodies and levels of IL-8, MIP-1α, MCP-1, MIP-1β and RANTES were measured using the respective cross-reacting human antibodies. Measurements were taken 14 days before the start of treatment and on day 55, when the monkeys were euthanized. The results are presented in Tables 167 and 168.

TABLE 167 Effect of antisense oligonucleotide treatment on cytokine/chemokine levels (pg/mL) in cynomolgus monkeys on day −14 IL-1β IL-6 IFN-γ TNF-α IL-8 MIP-1α MCP-1 MIP-1β RANTES PBS 350 3 314 32 82 27 277 8 297 ISIS 416850 215 1 115 4 45 14 434 31 4560 ISIS 449409 137 1 37 9 34 13 290 14 2471 ISIS 445522 188 5 172 16 32 22 297 27 3477 ISIS 449710 271 7 1115 72 29 20 409 18 1215 ISIS 449707 115 1 34 6 106 16 294 13 3014 ISIS 449711 79 2 29 6 156 20 264 24 3687 ISIS 449708 35 1 27 12 184 11 361 19 11666 ISIS 416858 103 0 32 4 224 11 328 37 6521 ISIS 445531 101 2 68 9 83 25 317 22 7825

TABLE 168 Effect of antisense oligonucleotide treatment on cytokine/chemokine levels (pg/mL) in cynomolgus monkeys on day 55 IL-1β IL-6 IFN-γ TNF-α IL-8 MIP-1α MCP-1 MIP-1β RANTES PBS 453 3 232 191 68 21 237 34 775 ISIS 416850 106 1 19 16 620 17 887 50 27503 ISIS 449409 181 0 25 8 254 17 507 47 8958 ISIS 445522 341 2 83 18 100 22 592 63 16154 ISIS 449710 286 2 176 26 348 27 474 53 22656 ISIS 449707 97 1 24 16 48 12 264 49 1193 ISIS 449711 146 7 22 31 110 17 469 91 3029 ISIS 449708 131 0 18 17 85 23 409 128 4561 ISIS 416858 28 1 9 15 167 11 512 47 5925 ISIS 445531 155 1 15 16 293 12 339 84 5935

Example 47 Measurement of Viscosity of ISIS Antisense Oligonucleotides Targeting Human Factor 11

The viscosity of antisense oligonucleotides targeting human Factor 11 was measured with the aim of screening out antisense oligonucleotides which have a viscosity more than 40 cP at a concentration of 165-185 mg/mL.

ISIS oligonucleotides (32-35 mg) were weighed into a glass vial, 120 μL of water was added and the antisense oligonucleotide was dissolved into solution by heating the vial at 50° C. Part of (75 μL) the pre-heated sample was pipetted to a micro-viscometer (Cambridge). The temperature of the micro-viscometter was set to 25° C. and the viscosity of the sample was measured. Another part (20 μL) of the pre-heated sample was pipetted into 10 mL of water for UV reading at 260 nM at 85° C. (Cary UV instrument). The results are presented in Table 169.

TABLE 169 Viscosity and concentration of ISIS antisense oligonucleotides targeting human Factor 11 Viscosity Concentration ISIS No. (cP) (mg/mL) 412223 8 163 412224 98 186 412225 >100 162 413481 23 144 413482 16. 172 416848 6 158 416850 67 152 416851 26 187 416852 29 169 416856 18 175 416858 10 166 416859 10 161 416860 >100 154 416861 14 110 416863 9 165 416866 >100 166 416867 8 168 445498 21 157 445504 20 139 445505 9 155 445509 >100 167 445513 34 167 445522 63 173 445522 58 174 445530 25 177 445531 15 155 445531 20 179 449707 7 166 449708 9 188 449709 65 171 449710 7 186 449711 6 209 451541 10 168 

1-54. (canceled)
 55. A method of treating a thromboembolic complication in an animal, comprising administering to an animal in need thereof a compound comprising a modified oligonucleotide consisting of 12 to 30 linked nucleosides, wherein the modified oligonucleotide is at least 90% complementary to a Factor 11 nucleic acid.
 56. The method of claim 55, wherein the administering prolongs aPTT in the animal.
 57. The method of claim 55, wherein the administering reduces platelet quantity as measured by platelet factor-4 quantification in the animal.
 58. The method of claim 55, wherein the administering reduces amount of bleeding in response to injury in the animal.
 59. The method of claim 55, wherein the administering prevents thrombus formation in the animal.
 60. The method of claim 55, wherein the thromboembolic complication is any of the group consisting of deep vein thrombosis, pulmonary embolism, myocardial infarction, and stroke.
 61. The method of claim 55, wherein the compound is for coadministation with any of the group consisting of aspirin, clopidogrel, dipyridamole, heparin, lepirudin, ticlopidine, warfarin, apixaban, rivaroxaban, LOVENOX, and Factor Xa inhibitor.
 62. The method of claim 55, wherein the compound is for concomitant administration with any of the group consisting of aspirin, clopidogrel, dipyridamole, heparin, lepirudin, ticlopidine, warfarin, apixaban, rivaroxaban, LOVENOX, and Factor Xa inhibitor.
 63. A compound, comprising a modified oligonucleotide consisting of 12 to 30 linked nucleosides having a nucleobase sequence comprising a portion of at least 8 contiguous nucleobases complementary to an equal length portion of nucleobases 1275 to 1318 of SEQ ID NO: 1, wherein the nucleobase sequence of the modified oligonucleotide is at least 90% complementary to SEQ ID NO:
 1. 64. The compound of claim 63, wherein the modified oligonucleotide is at least 95% complementary to SEQ ID NO:
 1. 65. The compound of claim 63, wherein the modified oligonucleotide is 100% complementary to SEQ ID NO:
 1. 66. The compound of claim 63, wherein the nucleobase sequence of the modified oligonucleotide comprises the sequence of SEQ ID NO: 217 or
 223. 67. The compound of claim 63, wherein the nucleobase sequence of the modified oligonucleotide consists of the sequence of SEQ ID NO: 217 or
 223. 68. The compound of claim 63, consisting of a single-stranded modified oligonucleotide.
 69. The compound of claim 68, wherein at least one internucleoside linkage is a modified internucleoside linkage.
 70. The compound of claim 69, wherein each internucleoside linkage is a phosphorothioate internucleoside linkage.
 71. The compound of claim 68, wherein at least one nucleoside comprises a modified sugar.
 72. The compound of claim 71, wherein at least one modified sugar is a bicyclic sugar.
 73. The compound of claim 72, wherein each of the at least one bicyclic sugar comprises a 4′-(CH₂)_(n)—O-2′ bridge, wherein n is 1 or
 2. 74. The compound of claim 72, wherein each of the at least one bicyclic sugar comprises a 4′-CH(CH₃)—O-2′ bridge.
 75. The compound of claim 71, wherein at least one modified sugar comprises a 2′-β-methoxyethyl group.
 76. The compound of claim 68, comprising at least one tetrahydropyran modified nucleoside wherein a tetrahydropyran ring replaces the furanose ring.
 77. The compound of claim 76, wherein each of the at least one tetrahydropyran modified nucleoside has the structure:

wherein Bx is an optionally protected heterocyclic base moiety.
 78. The compound of claim 68, wherein at least one nucleoside comprises a modified nucleobase.
 79. The compound of claim 78, wherein the modified nucleobase is a 5-methylcytosine.
 80. The compound of claim 68, wherein the modified oligonucleotide comprises: a gap segment consisting of linked deoxynucleosides; a 5′ wing segment consisting of linked nucleosides; a 3′ wing segment consisting of linked nucleosides; wherein the gap segment is positioned immediately adjacent to and between the 5′ wing segment and the 3′ wing segment, and wherein each nucleoside of each wing segment comprises a modified sugar.
 81. The compound of claim 80, wherein the modified oligonucleotide consists of 20 linked nucleosides.
 82. The compound of claim 81, wherein the modified oligonucleotide comprises: a gap segment consisting of ten linked deoxynucleosides; a 5′ wing segment consisting of five linked nucleosides; a 3′ wing segment consisting of five linked nucleosides; wherein the gap segment is positioned immediately adjacent and between the 5′ wing segment and the 3′ wing segment, wherein each nucleoside of each wing segment comprises a 2′-O-methoxyethyl sugar, wherein each internucleoside linkage is a phosphorothioate linkage, and wherein each cytosine is a 5-methylcytosine.
 83. A compound, comprising a modified oligonucleotide consisting of 12 to 30 linked nucleosides and having a nucleobase sequence comprising at least 12 contiguous nucleobases of SEQ ID NO: 217 or
 223. 84. The compound of claim 83, consisting of a single-stranded modified oligonucleotide.
 85. The compound of claim 84, wherein at least one internucleoside linkage is a modified internucleoside linkage.
 86. The compound of claim 85, wherein each internucleoside linkage is a phosphorothioate internucleoside linkage.
 87. The compound of claim 84, wherein at least one nucleoside comprises a modified sugar.
 88. The compound of claim 87, wherein at least one modified sugar is a bicyclic sugar.
 89. The compound of claim 88, wherein each of the at least one bicyclic sugar comprises a 4′-(CH₂)_(n)—O-2′ bridge, wherein n is 1 or
 2. 90. The compound of claim 88, wherein each of the at least one bicyclic sugar comprises a 4′-CH(CH₃)—O-2′ bridge.
 91. The compound of claim 87, wherein at least one modified sugar comprises a 2′-β-methoxyethyl group.
 92. The compound of claim 84, comprising at least one tetrahydropyran modified nucleoside wherein a tetrahydropyran ring replaces the furanose ring.
 93. The compound of claim 92, wherein each of the at least one tetrahydropyran modified nucleoside has the structure:

wherein Bx is an optionally protected heterocyclic base moiety.
 94. The compound of claim 84, wherein at least one nucleoside comprises a modified nucleobase.
 95. The compound of claim 94, wherein the modified nucleobase is a 5-methylcytosine.
 96. The compound of claim 84, wherein the modified oligonucleotide comprises: a gap segment consisting of linked deoxynucleosides; a 5′ wing segment consisting of linked nucleosides; a 3′ wing segment consisting of linked nucleosides; wherein the gap segment is positioned immediately adjacent to and between the 5′ wing segment and the 3′ wing segment, and wherein each nucleoside of each wing segment comprises a modified sugar.
 97. The compound of claim 96, wherein the modified oligonucleotide consists of 20 linked nucleosides.
 98. The compound of claim 97, wherein the modified oligonucleotide comprises: a gap segment consisting of ten linked deoxynucleosides; a 5′ wing segment consisting of five linked nucleosides; a 3′ wing segment consisting of five linked nucleosides; wherein the gap segment is positioned immediately adjacent and between the 5′ wing segment and the 3′ wing segment, wherein each nucleoside of each wing segment comprises a 2′-O-methoxyethyl sugar, wherein each internucleoside linkage is a phosphorothioate linkage, and wherein each cytosine is a 5-methylcytosine.
 99. The compound of claim 98, wherein the nucleobase sequence of the modified oligonucleotide comprises the sequence of SEQ ID NO: 217 or
 223. 100. The compound of claim 98, wherein the nucleobase sequence of the modified oligonucleotide consists of the sequence of SEQ ID NO: 217 or
 223. 101. The compound of claim 84, wherein the nucleobase sequence of the modified oligonucleotide comprises the sequence of SEQ ID NO: 217 or
 223. 102. The compound of claim 84, wherein the nucleobase sequence of the modified oligonucleotide consists of the sequence of SEQ ID NO: 217 or
 223. 103. A composition, comprising: a modified oligonucleotide consisting of 12 to 30 linked nucleosides and having a nucleobase sequence comprising a portion of at least 8 contiguous nucleobases complementary to an equal length portion of nucleobases 1275 to 1318 of SEQ ID NO: 1, wherein the nucleobase sequence of the modified oligonucleotide is at least 90% complementary to SEQ ID NO: 1, or a salt thereof; and a pharmaceutically acceptable carrier or diluent.
 104. The composition of claim 103 further comprising coadministation with any of the group consisting of aspirin, clopidogrel, dipyridamole, heparin, lepirudin, ticlopidine, warfarin, apixaban, rivaroxaban, LOVENOX, and Factor Xa inhibitor.
 105. The compound of claim 103 further comprising concomitant administration with any of the group consisting of aspirin, clopidogrel, dipyridamole, heparin, lepirudin, ticlopidine, warfarin, apixaban, rivaroxaban, LOVENOX, and Factor Xa inhibitor.
 106. A composition, comprising: a modified oligonucleotide consisting of 12 to 30 linked nucleosides and having a nucleobase sequence comprising at least 12 contiguous nucleobases of SEQ ID NO: 217 or 223, or a salt thereof; and a pharmaceutically acceptable carrier or diluent.
 107. A compound, comprising a modified oligonucleotide consisting of 12 to 30 linked nucleosides having a nucleobase sequence comprising a portion of at least 8 contiguous nucleobases complementary to an equal length portion of nucleobases 738 to 762 of SEQ ID NO: 1, wherein the nucleobase sequence of the modified oligonucleotide is at least 90% complementary to SEQ ID NO:
 1. 108. A compound, comprising a modified oligonucleotide consisting of 12 to 30 linked nucleosides having a nucleobase sequence comprising a portion of at least 8 contiguous nucleobases complementary to an equal length portion of nucleobases 656 to 704 of SEQ ID NO: 1, wherein the nucleobase sequence of the modified oligonucleotide is at least 90% complementary to SEQ ID NO:
 1. 109. A compound, comprising a modified oligonucleotide consisting of 12 to 30 linked nucleosides having a nucleobase sequence comprising a portion of at least 8 contiguous nucleobases complementary to an equal length portion of nucleobases 1018 to 1042 of SEQ ID NO: 1, wherein the nucleobase sequence of the modified oligonucleotide is at least 90% complementary to SEQ ID NO:
 1. 110. A compound, comprising a modified oligonucleotide consisting of 12 to 30 linked nucleosides having a nucleobase sequence comprising a portion of at least 8 contiguous nucleobases complementary to an equal length portion of nucleobases 1062 to 1091 of SEQ ID NO: 1, wherein the nucleobase sequence of the modified oligonucleotide is at least 90% complementary to SEQ ID NO:
 1. 111. A compound, comprising a modified oligonucleotide consisting of 12 to 30 linked nucleosides and having a nucleobase sequence comprising at least 12 contiguous nucleobases of any of the group consisting of SEQ ID NOs:15 to 216, 218-222 and 224 to
 269. 